Abstract: The present study characterized the effects of withdrawal from cocaine on the expression of NMDA receptor subunits (NR1, NR2B) and neuronal nitric oxide synthase. FosB induction was measured to confirm that repeated cocaine exposure influenced protein expression, as previously reported. Administration of cocaine followed by 24 h, 72 h, or 14 days of withdrawal resulted in alterations of NR1 and NR2B subunits and neuronal nitric oxide synthase expression as measured by immunohistochemical labeling of rat brain sections. Optical density analyses revealed significant up‐regulation of NR1 in the ventral tegmental area at 72 h and 14 days of withdrawal. Structure‐specific and withdrawal time‐dependent alterations in NR2B expression were also found. After 24 h of withdrawal, cocaine‐induced decreases in NR2B expression were observed in the nucleus accumbens shell, whereas increases in NR2B expression were found in medial cortical areas. Two weeks of withdrawal from cocaine caused an ∼50% increase in NR2B subunit expression in regions of the cortex, neostriatum, and nucleus accumbens. In contrast, cocaine‐induced up‐regulation of neuronal nitric oxide synthase was transient and evident in cortical areas only at 24 h after the last drug injection. The results suggest that region‐specific changes in interactions among proteins associated with the NMDA receptor complex may underlie neuronal adaptations following repeated cocaine administration.