Our laboratory has reported previously the characteristics of specific AVP binding to rat hippocampal synaptic membranes (SPM) in the presence of Ni2+ [Costantini MG, Pearlmutter AF: J Biol Chem 259: 11739-11745, 1984]. We extended our investigation to determine the effects of Ni2+, (AVP), and AVP analogs on SPM protein phosphorylation. Ni2+ (5 mM) caused a dramatic reduction in phosphorylation of most SPM phosphoproteins. The most prominent protein which is phosphorylated in SPM has a molecular weight of 48 kilodaltons (KDa) and has been named B50 or F1; this protein shows altered phosphorylation in vitro in response to long-term potentiation in vivo as well as changes induced by exposure of SPM to ACTH (1-24), dopamine, and somatostatin. AVP and related peptides reduced phosphorylation of this pre-synaptic phosphoprotein in the following order of potency: AVP = oxytocin greater than DG-AVP greater than dDAVP greater than d(CH2)5Tyr(Me)AVP = [pGlu4,Cyt6]AVP-(4-9). Except for the pressor antagonist d(CH2)5Tyr(Me)AVP, this corresponds to their relative efficacy in displacing 3H-AVP from high-affinity specific binding sites on rat hippocampal synaptic membranes. Ni2+ did not alter the degree of inhibition caused by the peptides. When SPM were treated with AVP after the attainment of maximum 32P incorporation, AVP inhibited dephosphorylation over a 30-min period. Our results show that AVP can alter both phosphorylation and dephosphorylation of hippocampal SPM phosphoproteins in vitro; the direction of these effects depends upon experimental conditions. Since B50/F1 is known to be a substrate for protein kinase C, AVP may act by inhibition of protein kinase C activity, either directly or indirectly.