Dictyostelium discoideum spores can be activated to initiate germination either endogenously via a diffusible autoactivator, or exogenously via heat. Following activation, three successive stages of germination occur, the lag stage, spore swelling and amoebal emergence. A previous study [Lydan M. A. and Cotter D. A. (1994) FEBS Lett. 115, 137-142] has shown that spore swelling is dependent on the activity of calmodulin. In this study, the calmodulin antagonists trifluoperazine and calmidazolium inhibited autoactivation, but had no effect upon heat activation. These agents also inhibited amoebal emergence following either form of activation. The effects caused by the anti-calmodulin agents were specific to an inhibition of calmodulin function since agents which modulate the activity of protein kinase C had no effect upon spore germination. A calcium-dependent calmodulin-binding protein of about 64,000 M(r) may be associated with the process of autoactivation since it was only seen in those spores which respond to the autoactivator. Overall, this study provides evidence to show that calmodulin plays a regulatory role during autoactivation and amoebal emergence during spore germination in D. discoideum and provides evidence for the calmodulin-dependent mechanisms which mediate each of these phases of germination.