We have identified a homologue (ponB) of the ponticulin gene (ponA), an F-actin binding protein, in the expressed sequence tag library generated to mRNA isolated from fusion-competent cells of Dictyostelium discoideum. PonB is predicted to have many of the same characteristics as ponticulin. Both proteins are predicted to possess a cleaved signal peptide, a glycosyl anchor, an amphipathic beta-strand structure and six conserved cysteines. Because of the sequence similarity and predicted conserved structures, this gene constitutes the second member of a ponticulin gene family. Unlike ponticulin, ponB is not expressed in axenically grown cells or during the asexual reproductive phase of D. discoideum. PonB is expressed by cells grown on bacterial lawns and by cells induced to be fusion-competent, i.e., gametes. The expression of ponB correlates with the appearance of a new F-actin binding activity in cell lysates of bacterially grown ponA(-) cells. By immunofluorescence microscopy, ponB appears to be localized to vesicles and to the plasma membrane of bacterially grown cells. Because ponticulin is the major high-affinity link between the plasma membrane and the cytoskeleton, the ponticulin gene family is likely to be part of the redundant system of proteins involved in connecting the cytoskeleton to the plasma membrane.