We found previously that neural responses to ethanol and the dopamine D2 receptor (D2) agonist 2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) involve both epsilon protein kinase C (epsilonPKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanol- and D2-mediated activation of epsilonPKC and the relationship to PKA activation. In the present study, we used a new epsilonPKC antibody, 14E6, that selectively recognized active epsilonPKC when not bound to its anchoring protein epsilonRACK (receptor for activated C-kinase), and PKC isozyme-selective inhibitors and activators to measure PKC translocation and catalytic activity. We show here that ethanol and NPA activated epsilonPKC and induced translocation of both epsilonPKC and its anchoring protein, epsilonRACK to a new cytosolic site. The selective epsilonPKC agonist, pseudo-epsilonRACK, activated epsilonPKC but did not cause translocation of the epsilonPKC/epsilonRACK complex to the cytosol. These data suggest a step-wise activation and translocation of epsilonPKC after NPA or ethanol treatment, where epsilonPKC first translocates and binds to its RACK and subsequently the epsilonPKC/epsilonRACK complex translocates to a new subcellular site. Direct activation of PKA by adenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS), prostaglandin E1, or the adenosine A2A receptor is sufficient to cause epsilonPKC translocation to the cytosolic compartment in a process that is dependent on PLC activation and requires PKA activity. These data demonstrate a novel cross-talk mechanism between epsilonPKC and PKA signaling systems. PKA and PKC signaling have been implicated in alcohol rewarding properties in the mesolimbic dopamine system. Cross-talk between PKA and PKC may underlie some of the behaviors associated with alcoholism.