An extensive body of literature provides evidence for sexual dimorphism in drug abuse pattern, behavioral activation, and neurochemical levels. Female rats display a greater behavioral locomotor response to acute cocaine administration, condition faster and at lower doses, and also show greater hypothalamic-pituitary adrenal axis (HPA) activation and reward effects. However, the mechanisms underlying the sexually dimorphic responses to cocaine remain unclear. One biological basis for the observed gender differences in the behavioral response to cocaine may be hormonal fluctuations during the menstrual/estrous cycle; hormonal fluctuations during the estrous cycle have been shown to affect the response to cocaine. To date, no study has addressed which cellular mechanisms may affect the responses to cocaine in females. Both acute and chronic cocaine-induced stimulation of the dopaminergic system has been shown to affect various levels of DARPP-32/PP-1 and PKA/CREB pathways. Our hypothesis is observed gender differences in the behavioral response to cocaine may, in part, be regulated by differential activation of proteins in DARPP-32/PP1 or PKA/CREB pathways. We postulate that protein activation in PKA-mediated pathways may be sexually dimorphic and/or differentially activated by the estrous cycle. Because of the crucial role of ovarian hormones on female biological responses, a correlate of this hypothesis is that estrogen and progesterone differentially alter cocaine intracellular activation. In the NAc, cocaine-induced sex differences were observed in PKA-mediated intracellular cascades: males had higher PKA protein levels than females, while females had higher P-Thr-34-DARPP-32 protein levels than males. Cocaine-induced estrous cycle differences were observed in PKA, pCREB, PP-2B, P-Thr-34-DARPP-32 protein levels: 15 min after acute cocaine, PP-2B (CNA1) protein levels increased in estrus females, while PKA and pCREB levels decreased in estrus females. One hour after cocaine administration, P-Thr-34-DARPP-32 protein levels increased in the NAc of estrus and diestrus females, and in the CPu of diestrus females. Moreover, region-specific estrous cycle differences in P-Thr-34-DARPP-32 protein levels were observed: estrus females had higher accumbal P-Thr-34-DARPP-32 protein levels than diestrus females, whereas, in the CPu, diestrus females had higher P-Thr-34-DARPP-32 protein levels than estrus. Furthermore, cocaine-induced hormone replacement differences were observed in PKA, pCREB, and CFosB protein levels: accumbal PKA protein levels increased in vehicle and E+P co-administration group, while accumbal pCREB protein levels increased in vehicle and decreased in E+P co-administration group. Additionally, CFosB protein levels decreased in vehicle females. Moreover, when analyzed to their respective saline controls, accumbal PKA and pCREB protein levels were higher in the vehicle group when compared to hormone replacement groups and E+P co-administration group had lower pCREB protein levels compared to all other groups. Understanding how the estrous cycle and gonadal hormones affect cocaine-induced intracellular changes should allow for more efficient treatment for female addicts, which represent one-third of the current cocaine users.