OBJECTIVE: To obtain two-dimensional gel electrophoresis maps of the prefrontal cortex (PFC) proteins of normal and heroin-addicted rats for identifying the differentially expressed proteins in the addicted rats. METHODS: Rat models of heroin addiction were established, and the proteins in the PFC underwent two-dimensional gel electrophoresis with immobiline pH gradient isoelectric focusing as the first and vertical SDS-PAGE as the second dimension. ImageMaster 2D 5.0 analysis software, matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) or MALDI-TOF-TOF-MS as well as NCBInr database searching were performed to separate and identify the proteome in the rat PFC. RESULTS: Seven protein spots to represent 5 proteins were identified by PMF and MALDI-TOF-TOF-MS. Among those proteins, glucose-regulated protein (58 kD) and 26 S proteasome subunit p40.5 existed only in heroin-addicted rats, in which ATP synthase D chain was down-regulated. Ndufa10 and Eno1 were present in both groups, but their molecular mass and pI were different. CONCLUSION: Immobilized pH gradient two-dimensional gel electrophoresis allows good reproducibility in separation and identification of the proteome in the PDF of heroin-addicted rats, which facilitates further investigation of pathogenic mechanisms of heroin addiction and central nerve injury.