To determine whether chronic oxytocin pretreatment inhibits adenylyl cyclase, we compared adenylyl cyclase activity in membranes prepared from cultured, immortalized rat myometrial cells that were untreated or pretreated for 24 h with oxytocin. Chronic oxytocin pretreatment (1 × 10−5 M for 24 h) attenuated basal, guanosine triphosphate (1 × 10−5 M)-, isoproterenol (1 × 10−4 M)-, forskolin (1 × 10−5 M)-, MnCl2 (20 mM)- or NaF (1 × 10−2 M)-stimulated adenylyl cyclase activity by 27 ± 5% to 39 ± 11% (n = 6, p < 0.05). Oxytocin pretreatment for 2 h (n = 5) did not produce a significant effect. To understand the mechanism by which oxytocin pretreatment decreased activity of the adenylyl cyclase pathway, we compared effects of pretreatment with either oxytocin or phenylephrine on adenylyl cyclase activity and determined the effects of Gi inhibition and protein kinase C (PKC) depletion. Chronic (24 h) phenylephrine pretreatment (1 × 10−4 M) had effects similar to those of oxytocin pretreatment (1 × 10−5 M). PKC depletion with phorbol 12-myristate 13-acetate (1 × 10−6 M, 41 h) prevented attenuation of adenylyl cyclase activity by oxytocin pretreatment (1 × 10−5 M for 24 h). Inhibition of Gi by pertussis toxin pretreatment (1.25 μg/ml, 41 h) had no significant effect. These findings suggest that chronic oxytocin pretreatment desensitizes the adenylyl cyclase pathway by a cross-regulatory mechanism that involves activation of Gq and PKC.