Although the cAMP and phosphoinositide (PI) second messenger systems are involved in olfactory signal transduction, aspects of their roles remain unclear. We have further examined the rapid kinetics of cAMP fluctuations in response to odorants in rat olfactory cilia isolated by calcium shock. Odorants cause a rapid and transient subsecond elevation of cAMP levels, as well as a more sustained signal lasting 5–10 sec. Basal cAMP levels demonstrate a biphasic calcium dependence; calcium enhanced both adenylyl cyclase (AC) and phosphodiesterase (PDE) activities. The odorant-induced cAMP response also demonstrated a biphasic dependence on calcium, with peak activity at 10 microM free calcium. All odorants tested were found to stimulate cAMP accumulation, and the dose-response curves were multiphasic, with less stimulation seen at higher concentrations. Dose-response curves performed for isovaleric acid at two free calcium concentrations demonstrated that calcium can influence cellular responsiveness to odorants and may be involved with signal potentiation as well as desensitization.