In vitro studies were conducted to evaluate the possible mechanisms of LH-induced desensitization of the rabbit follicular adenylyl cyclase (AC) system. We tested the effects of cAMP, dibutyryl cAMP, and inhibitors of various cellular functions on LH-stimulated AC activity as well as the reversibility of AC desensitization. Refractoriness of the AC to LH was induced by a1 1- or 2-h incubation of Graafian follicles with 10 microgram/ml LH. We fund that the initial 60-min phase of AC desensitization to LH in Graafian follicles was not prevented by a 60-min preincubation of follicles with 11 microM puromycin, 30 microM cycloheximide, 8 microM actinomycin D, 5 microgram/ml cytochalasin B, 50 microM colchicine, or 1 or 10 mM trinitrophenol or by a 90-min preincubation of follicles with 50 microM colchicine. We evaluated the effects of cAMP and dibutyryl cAMP on LH-stimulated AC activity by incubating Graafian follicles with these nucleotides for 30-min to 4 h. While LH-stimulated AC activity was not significantly reduced in follicles which had been incubated 30-min or 1-h with either nucleotide, 2 h incubations resulted in significant reductions in LH-stimulated AC activity, and 4-h incubations promoted a complete refractoriness of the LH-stimulable AC. cAMP also caused desensitization of the FSH-stimulable AC in 4-h incubations, but not in the 1-h incubations. Lastly, once the follicular AC was desensitized to LH, neither 4-guanylyl imidodiphosphate nor ATP could reverse desensitization. These results indicate that AC desensitization in rabbit Graafian follicles is biphasic event. The initial 60-min phase is not mediated by cAMP, RNA, or protein synthetic events, by energy-requiring events inhibited by trinitrophenol, or by microtubule- or micro-filament-associated processes. A secondary phase occurs within 2-h and appears to be mediated, at least in part, by cAMP.