Habituation is an important behavioural response to repeated exposure of painful stimuli¹. In this study, we investigated habituation related changes of brain activation with respect to repeated painful heat stimuli in four different pain processing brain regions^{2, 3} using BOLD-fMRI.Four healthy volunteers (1f/3m, one subject investigated twice, 32 ± 9 years) were measured on a clinical whole-body 3 T MR scanner (Magnetom PRISMA, Siemens, Erlangen, Germany) with a vendor supplied 64-channel head matrix coil. Functional imaging data where acquired using a standard GRE-EPI sequence (in plane resolution 2.1 mm, TA = 11min, TE = 30 ms, three respectively two experiments with TR = 2 s and 1.6 s; slice thickness 2.6 mm and 3.2 mm, each with a gap of 20 %). The fMRI protocol consisted of 20 alternating 16 s STIM and 16 s REST periods. During STIM, three heat pulses at an individually adjusted temperature were applied to the back of the right hand (VAS = 5⁴, T_max,1s = 51 °C, T_max,2s = T_max,1s – 2 °C , T_max,3s = T_max,1s – 2.5 °C, interstimulus interval: 3 s at baseline temperature of 32 °C). During rest the temperature was hold constant at baseline temperature. Acquired EPI series were analysed using SPM8 software package (http://www.fil.ion.ucl. ac.uk/spm, motion correction, slice-time correction, smoothing). To determine temporal BOLD signal variations the data were stepwise processed by shifting a time window of 4.27 min with step size of 32 s, resulting in 12 different windows. Applying a general linear model provided statistical parametric maps for the STIM vs. REST contrast coefficient and for the constant term in the regression for each temporal step. ROI masks were defined in MNI space for four brain regions (left and right insula (rIns, lIns), anterior cingulate cortex (ACC) and precuneus) and transformed into each EPI dataset to calculate the time course of the mean BOLD-fMRI amplitude in the selected brain regions. The latter was calculated in percentage by dividing voxelwise the STIM vs. REST contrast coefficient by the constant regression term⁵. Stimuli related activation of brain regions was assumed, if, first, more than 10 % of the voxels of the corresponding ROI were significantly activated (N_voxel, p < 0.05) for at least one time window and, second, the mean beta value per person and ROI was positive, depicting a positive BOLD response, respectively. Additionally, the time course of the number of N_voxel was correlated with the time course of the BOLD amplitude for each ROI and each volunteer, respectively.All volunteers showed pain stimuli related brain activation in lIns and rIns, four in ACC and two in precuneus. The range of mean BOLD amplitude values for activated brain regions for the first time window is listed in Tab. 1. The relative BOLD-fMRI amplitude changes over the duration of nociception are shown in Fig. 2. lIns and rIns revealed smaller variations compared to ACC and precuneus in all subjects. Furthermore, the correlation between the time courses of BOLD amplitude and N_voxel was significant (p < 0.05) for all ROIs (except for one volunteer in rIns and precuneus), supporting that BOLD signal changes are related to functional habituation, and are not caused by e.g. single random high activated voxels within the ROIs. Reliable region specific brain activation and changes during prolonged noxious stimulation should be considered in further acute pain studies, in particular, if long measurement times are required as, e.g., in MR spectroscopy