In addition to its expression on subsets of axons, grasshopper Semaphorin I (Sema I, previously called Fasciclin [Fas] IV) is expressed on an epithelial stripe in the limb bud, where it functions in the guidance of two sensory growth cones as they abruptly turn upon encountering this sema I boundary. We report here on the cloning and characterization of two sema genes in Drosophila, one in human, and the identification of two related viral sequences, all of which encode proteins with conserved Semaphorin domains. Drosophila sema (D-Sema) I is a transmembrane protein, while D-Sema II and human Sema III are putative secreted proteins that are similar to the recently reported chick collapsin. D-Sema I and D-Sema II are expressed by subsets of neurons and muscles. Genetic analysis in Drosophila reveals that semall is an essential gene that is required for both proper adult behavior and survival.