The semaphorins are a large, evolutionarily conserved family of signaling molecules with broad functions during development. The class 3 semaphorins are a subclass of secreted semaphorins found in vertebrates. There have been six class 3 semaphorins identified to date (sema3A to sema3F) and some have been shown to function in axon guidance and cardiovascular development. However, the functions of many class 3 semaphorins and their potential interactions in vivo are still not well understood. As a step toward understanding the actions of all class 3 semaphorins in vivo, we have cloned and analyzed the developmental expression pattern of a novel zebrafish class 3 semaphorin, sema3H [corrected] sema3H [corrected] is expressed in a dynamic pattern throughout the first 3 days of development. It is expressed in the adaxial cells of the somite during somitogenesis. In the brain, sema3H [corrected] is expressed in cell clusters in the midbrain and diencephalon, and is expressed in the telencephalon in close proximity to the olfactory epithelium. sema3H [corrected] also is expressed in the pharyngeal arches, the pectoral fin bud, and the developing pronephros. These results provide a basis for studying how expression of multiple semaphorins could be essential for aspects of early development.