Lysozymes, especially c-type lysozymes, are well-recognized bacteriolytic factors widely distributed in the animal kingdom and play a mainly protective role in host defense. The relatives of c-type lysozymes, alpha-lactalbumins, however, are only found in mammalian milk and possess a distinct biological function. These two proteins, having similar amino acid sequences, gene structure, and dimensional conformation, belong to the c-type lysozyme/alpha-lactalbumin family. Using human lysozyme as an information probe, we cloned four human cDNAs encoding homologues of human lysozyme; these were named LYZL2, LYZL4, LYZL6, and SPACA3 by the HUGO Gene Nomenclature Committee. Of these four, SPACA3 has been reported to code an intra-acrosomal sperm protein SLLP1. To our knowledge, the other three are reported here for the first time. Using Northern blot hybridization, including 16 different human tissues, we found that these four lysozyme-like genes were all highly expressed in the testis/epididymis. Further analysis of one, LYZL4, by in situ hybridization revealed that its mRNA was only detected in the epithelium of human epididymis, most abundantly in the caput, suggesting that LYZL4 plays a physiological role in male reproduction. By sequence analysis, we found that two essential catalytic residues of the human lysozyme were conserved in LYZL2 and LYZL6, whereas one site in LYZL4 and two sites in SPACA3 were replaced. The LYZL2, LYZL4, LYZL6, and SPACA3 genes were mapped to human chromosome 10p11.23, 3p21.33, 17q11.2, and 17q12, respectively, and displayed a similar genomic structure. Our data suggest that these four lysozyme-like genes, which have arisen from a common progenitor gene, play a major role in human reproduction.