Zebrafish embryos have three or four identifiable primary motoneurons per hemisegment. We previously reported that, while several ventral cells initially express the zebrafish Islet-1 (Isl-1) gene, a member of the LIM/homeobox gene family, the expression of this gene becomes restricted to a single or a pair of cells slightly anterior to each segment border by 16 hr after fertilization. Double staining by in situ hybridization and immunohistochemistry strongly suggested that these cells were mainly rostral primary motoneurons. Here, we have isolated two novel zebrafish cDNA clones for more Isl-1 family genes, termed zfIsl-2 and zfIsl-3. zfIsl-2 mRNA starts to be expressed in the ventral midsegmental cells per hemisegment around 15 hr. Double labeling experiments have shown that these midsegmental cells are the caudal primary motoneuron (CaP) and its variant equivalence pair. Our results revealed the heterogeneity in the expressed genes among primary motoneurons before the fates of the primary motoneurons are irreversibly determined, and further suggest the involvement of the Isl-1 and zfIsl-2 genes in the determination of cellular identities by primary motoneurons in embryonic zebrafish. zfIsl-3 mRNA is not expressed in motoneurons but is expressed at 17 hr, mainly in the ventral myotomes. This suggests that zfIsl-3 may be involved in the regional specification of the myotome and also in target recognition by CaP. zfIsl-2 is also expressed throughout the developing eye and tectal region of the midbrain, the target for the retinal axons. In the ventral spinal cord of the spadetail mutant embryo, which has defects in the somites, the cells expressing zfIsl-2 mRNA significantly decreased in number in contrast to the increase in cells expressing Isl-1 mRNA, suggesting the influence of the somites on the expression of both genes.