We report here the molecular cloning and characterization of a novel human actin capping protein alpha3 (cpalpha3) cDNA, an orthologue of the mouse male germ cell-specific cpalpha3, and the organization of the human cpalpha3 genomic structure. The entire coding region of the human cpalpha3 cDNA showed 82.1% similarity with the mouse cpalpha3. The predicted amino acid sequence was 91.3% identical to the mouse protein and the actin-binding motif in the C-terminal region is highly conserved among species. The mRNA of the human cpalpha3 gene was found to be exclusively expressed in the testis. Western blot analysis detected a 33 kDa protein in human testis and sperm. Immunohistochemistry showed that the main localization of human CPalpha3 protein was in the neck region of ejaculated sperm, with moderate and faint signals also detected in the tail and postacrosome region respectively. Furthermore the localization of CPalpha3 coincided with the species-specific distribution of actin in human sperm. The human cpalpha3 gene was mapped to chromosome 12p12 by computer database cloning of human genomic DNA and was proven to be intronless. CPalpha3 may play a physiologically important role in sperm architecture as well as in fertility of the human male.