Evolution of the egg-laying hormone (ELH) gene family was examined in the genus Aplysia using genomic Southern blotting, gene cloning, and immunocytochemical techniques to identify and characterize homologous sequences. Most of the species examined have fewer than the 4-5 ELH-related genes present in the A. californica genome (Mahon et al., 1985; Scheller et al., 1983). In A. parvula there are 2 ELH genes, and unlike A. californica, no sequences were found to encode the A or B peptides. The 2 A. parvula ELH genes share at least 90% DNA sequence homology, while the homology between the A. parvula and A. californica ELH genes is 71%. The structural organization of the A. parvula ELH precursor is quite similar to the ELH precursor of A. californica, with all but one of the potential proteolytic cleavage sites conserved. The overall amino acid homology between the A. parvula and A. californica ELH precursors is 66%; however, the alpha and beta bag cell peptides, as well as ELH, are more highly conserved, suggesting that these peptides have important physiological and behavioral roles within both Aplysia species. Immunocytochemical studies indicate that the A. parvula ELH genes are expressed in 2 bag-cell-like clusters of about 40 neurons each in the abdominal ganglion. There does not appear to be an atrial gland in A. parvula; however, ELH-immunoreactive peripheral neurons and their processes are observed along the perimeter of the large hermaphroditic duct.