The semaphorin family contains secreted and transmembrane signaling proteins that function in the nervous, immune, and cardiovascular systems. Chick collapsin-1 is a repellent for specific growth cones. Two other secreted members of the semaphorin family, collapsin-2 and -3, are structurally similar to collapsin-1 but have different biological activities. Semaphorins contain a 500 amino acid family signature semaphorin domain. We show in this study that (1) the semaphorin domain of collapsin-1 is both necessary and sufficient for biological activity, (2) the semaphorin domain contains a 70 amino acid region that specifies the biological activity of the three family members, and (3) the positively charged carboxy terminus potentiates activity without affecting specificity. We propose that semaphorins interact with their receptors through two independent binding sites: one that mediates the biological response and one that potentiates it.