Relaxin, a uterine relaxant secreted by the corpus luteum, was able to elevate cAMP concentrations in the presence of 1-methyl-3-isobutyl xanthine (MIX) (0.1 mM) or forskolin (0.4 microM) in a time- and dose-dependent manner in rat myometrial cells in culture but not in stromal cells. The optimal culture conditions for the cAMP response were determined to be an initial plating density of 1-1.5 X 10(6) cells/ml (3 ml/35-mm dish) and a 2-day culture period. In the presence of MIX, the time course of cAMP elevation in response to relaxin exhibited a lag phase of more than 5 min before cAMP concentrations rose significantly. However, in the presence of forskolin, relaxin elevated cAMP within 1 min. The concentration-response relationships were almost identical in the presence of MIX or forskolin. Isoproterenol was able to increase cAMP concentrations in myometrial cultures in both the absence and presence of MIX and to elevate cAMP levels rapidly within 1 min. These data suggest that cAMP could play some role in the initiation of uterine relaxation mediated by relaxin.