Morphine has been used as a potent analgesic, having a high propensity to induce tolerance and physical dependence following their repeated administration. Although the mechanisms that underlie the development of dependence on morphine remain unclear, previous studies suggested that phosphorylations of diverse types of cellular proteins are crucial determinants of the neuroadaptive mechanisms associated with morphine dependence. Thus, understanding global phosphorylation events induced by chronic morphine administration is essential for understanding the complex signaling mechanisms of morphine dependence. This study characterized the alteration of tyrosine phosphorylation of frontal cortical proteins in morphine-dependent rat brains using a proteomic approach. Dependence was produced by continuous intracerebroventricular (i.c.v.) infusion of morphine (26 nmol/microl/h) for 72 h via osmotic minipumps in rats. Phosphotyrosyl (p-Tyr) protein spots in brain frontal cortical regions were detected by two-dimensional electrophoresis (2-DE) and immunoblotting with anti-p-Tyr-specific antibodies. The protein spots showing significant changes in tyrosine phosphorylation were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Similar patterns of protein expression were detected by 2-DE gels in morphine-dependent and saline-treated control rat brains. However, phosphotyrosine 2-DE images of the frontal cortical proteins from saline-treated control and morphine-dependent rat brains were apparently different. The densities of most matched p-Tyr protein spots were increased in morphine-dependent rat brains compared with that of control samples. Additional p-Tyr protein spots were detected in 2-DE image of morphine-dependent rat brains. Fifty of p-Tyr protein spots, corresponding to 40 different proteins, were identified from 2-DE gels of morphine-dependent rat brains. The identified proteins include enzymes, cytoskeletal proteins, cell signaling molecules, and other proteins. In conclusion, the first available phosphotyrosine proteomic resources of morphine dependence were established using an animal model. The findings illustrate the potential of proteomics as an effective technique for studying phosphorylation events of morphine dependence in brains.