Although a series of proteins in the brain have been shown to be qualitatively or quantitatively dysregulated following morphine administration, a systematic proteomic study has not been carried out so far. We therefore aimed to show the effect of morphine on protein levels in the rat brain. For this purpose rats were given a morphine base in subcutaneously placed pellets and subsequently the cerebral cortex, hippocampus and striatum were taken for proteomic studies after three days. Extracted proteins were run on two-dimensional gel electrophoresis, scanned and quantified by specific software. Proteins with significantly different levels were analysed by mass spectrometry (MALDI-TOF-TOF). Twenty-six proteins were found to be differentially expressed and were unambiguously identified. Dysregulated proteins were from several protein pathways and cascades including signaling, metabolic, protein handling, antioxidant and miscellaneous classes. These findings represent an initial approach to the generation of a 'morphinome' and may form the basis for further protein chemical studies as a valuable analytical tool. Moreover, the study reveals morphine-regulated proteins in different brain areas and indicates the pathways involved following morphine administration in the rat, the main species for pharmacological studies in the field.