In view of the potential impact of pituicyte morphology on neurohypophysial hormone secretion, we have studied the mechanisms involved in the shape changes induced by vasopressin (AVP) and oxytocin (OXT) in cultured rat pituicytes. Pituicytes induced to become stellate in the presence of 10 micro m adenosine revert to their nonstellate shape approximately 20 min after application of AVP or OXT. The IC50 for this effect is 0.1 nm for AVP and 36 nm for OXT. Both agonists induce Ca2+ signals in pituicytes, comprised of a transient peak and a plateau phase that is dependent on the presence of extracellular Ca2+. The EC50 values of AVP for the transient and sustained responses are 4.5 and 0.1 nm, respectively; corresponding values for OXT are 180 and 107 nm. We determined pharmacologically that these hormone-induced Ca2+ signals are mediated by the V1a subtype of vasopressin receptors, similar to what we previously observed for hormone-induced reversal of stellation. Removal of extracellular Ca2+ or chelation of intracellular Ca2+ partially prevented AVP from reversing stellation, suggesting a role for Ca2+ in this event. We previously established that adenosine-induced stellation of pituicytes occurs via RhoA inhibition. However, pharmacological experiments and pull-down assays presented here show that AVP-induced reversal of stellation does not involve RhoA activation. Rather, AVP was found to induce a time-dependent activation of Cdc42, another small GTPase involved in cytoskeletal plasticity. Activation of Cdc42 by AVP is sensitive to intra- and extracellular Ca2+ depletion, similar to AVP-induced reversal of stellation. Furthermore, AVP-induced reversal of stellation is blocked by expression of an NWASP fragment known to inhibit endogenous Cdc42.