Both AVP and dDAVP effect a transient increase in cytosolic free calcium (iCa2+) in cortical collecting tubule (CCT) cells. To investigate the physiological role of this increase in iCa2+, we examined the effect of TMB-8, a putative inhibitor of iCa2+ release, on the initial and sustained phase of AVP- and dDAVP-stimulated water permeability (Pf) in isolated, perfused CCTs. Pretreatment of tubules with TMB-8, 50 microM, suppressed the increase in osmotic water permeability (Pf) induced by 10 microU/ml AVP and dDAVP, but had no effect on the sustained phase of the response. When increased to 100 microM. TMB-8 inhibited the sustained phase of AVP action. A similar pattern was observed on AVP-stimulated adenyly cyclase activity in rabbit renal membranes. Pretreatment of tubules with 50 microM TMB-8 attenuated the initial increase in Pf in response to cholera toxin but not to 8-Br-cAMP or forskolin. There was no effect of this concentration of TMB-8 on the sustained phase of these agonists. These studies suggest that, in lower concentrations, TMB-8 inhibits the mobilization of iCa2+, which is important for the interaction of Gs with the catalytic unit of adenylyl cyclase and the initial increase in AVP-stimulated Pf. In higher concentrations, TMB-8 inhibits adenylyl cyclase activity directly.