The calcium-dependent regulatory protein calmodulin (CaM) mediates diverse cellular functions via a large number of calmodulin-binding and -dependent proteins (CaMBPs). The use of [35S]calmodulin, labeled during its expression (VU-1-CaM) in Escherichia coli, visualized over 25 CaMBPs in Dictyostelium discoideum. Seven, with M(r)s of 155,000, 91,000, 85,000, 48,000, 46,000, 38,000, and 28,000, were present only during sexual development. In addition, intracellular calmodulin levels were low during gamete formation but rose during cell fusion in response to the presence of extracellular calcium. Thus, calmodulin appears to mediate gamete formation and fusion through two distinct mechanisms: first, via unique developmentally regulated CaMBPs, and, second, via the regulation of intracellular calmodulin levels. The identification of the CaMBP spectrin in sexually developing Dictyostelium cells suggests that this cytoskeleton/plasma membrane, crosslinking protein may function during biomembrane fusion in D. discoideum as it does in other organisms.