Ovarian follicles of Hyalophora cecropia stopped accumulating [35S]vitellogenin when incubated in pertussis toxin, a Gi protein inactivator. At a cellular level, the responses to pertusis toxin resembled those described earlier to cell-permeant analogs of cyclic AMP. They included accelerated 36Cl-exchange, 86Rb+ uptake, and follicle cell swelling, which in turn resulted in a loss of epithelial patency. A 34% rise in follicular cAMP content accompanied these changes. In particulate fractions of follicle homogenates, pertussis toxin catalyzed the ADP-ribosylation of a polypeptide that resolved at 39 kDa in SDS-PAGE; rabbit antibodies to a C-terminal decapeptide common to 39 kDa mammalian Gi alpha-3 and G(o) alpha were bound in immunoblots at this same location. The findings suggest that a pertussis toxin-sensitive G alpha facilitates epithelial patency during vitellogenesis by suppressing cAMP levels. When follicles are released from this restraint, either experimentally with pertussis toxin or by progressing to the next phase in their normal program of development, cAMP levels rise and vitellogenesis terminates.