During early sexual development in Dictyostelium discoideum cell and pronuclear fusion are negatively regulated by an endogenous autoinhibitor. Here, the autoinhibitor was partially purified from the culture medium and found to inhibit both cell and pronuclear fusion while augmenting gamete numbers. These developmental effects suggested that calmodulin might be an intracellular target for the autoinhibitor. In support of this data, the partially purified autoinhibitor inhibited the calmodulin-dependent activation of phosphodiesterase in a dose-dependent manner, but had no effect on either a calmodulin-insensitive form of phosphodiesterase or the calmodulin-independent enzymes acid and alkaline phosphatase. Thus, the autoinhibitor of sexual development in Dictyostelium discoideum appears to regulate cell and pronuclear fusion at least in part by a direct effect on calmodulin.