The present study examines the effects of the antidepressant venlafaxine, a dual amine reuptake inhibitor, on (a) in vivo regulation of the densities of high- and low-affinity dihydroalprenolol (DHA) binding sites in the cortex of normal and reserpinized Sprague-Dawley rats and (b) targets beyond the beta adrenoceptor. While venlafaxine (30 mg/kg i.p. b.i.d.) administered for 4 d did not alter the DHA-binding parameters in the cortex of normal rats, it significantly reduced, in reserpinized animals, the number of up-regulated low-affinity sites (R(L)) which have been tentatively identified as serotonin(1B) sites. The drug did not influence the up-regulated high-affinity (R(H)) DHA-binding sites (beta-adrenoceptor sites). Venlafaxine failed to alter the up-regulated R(L) sites in brains of rats depleted of serotonin (5-HT) by p-chlorophenylalanine (PCPA) indicating that the normalization by venlafaxine of the up-regulated R(L) receptor population is mediated by increased synaptic 5-HT. Venlafaxine, given for a short period of time, thus mimicked the action of fluoxetine. While venlafaxine (20 mg/kg i.p. b.i.d.) given for 10 d did not change protein kinase A activity as assessed by the phosphorylation of kemptide in the 900 g supernatant or particulate fractions, the drug significantly reduced phosphorylated cAMP response-element binding protein (CREB-P) in nuclear lysates of cortex after chronic but not acute administration. Depletion of 5-HT by PCPA did not alter the venlafaxine-induced change in nuclear CREB-P. Lastly, analysis of reverse transcribed cortical CREB mRNA by competitive PCR indicated that the mean steady-state levels of CREB mRNA in venlafaxine vs. saline-treated animals were not significantly different. Therefore, since the phosphorylation status of CREB determines its transcriptional activity the reduction of nuclear CREB-P may be venlafaxine's most relevant action beyond the adrenoceptor.