An extensive analysis of testis-expressed genes in the ascidian Ciona intestinalis explored a large number of genes of unknown function. Here we characterized these genes or gene products in a multidimensional manner. We analyzed genes both highly and uniquely expressed in the testis, as expected from the EST analysis. Immunolocalization of these proteins revealed that they are all expressed in sperm. Sperm membrane/matrix proteins play essential roles in cell responses and intracellular signaling at fertilization. By immunoscreening with antisera against the detergent-soluble and membrane fractions of sperm, we isolated 49 potential cDNA clones for membrane/matrix proteins. These included several unidentified genes, including a protein with sequence similarity to mammalian testicular cancer antigen Sp17. These data should facilitate exploration of the functions of uncharacterized sperm proteins and ultimately elucidate new molecular mechanisms in sperm physiology.