Mammalian spermatogenesis is a highly ordered process that occurs in mitotic, meiotic, and postmeiotic phases. The unique mechanisms responsible for this tightly regulated developmental process suggest the presence of an intrinsic genetic program composed of spermatogenic cell-specific genes. In this study, we analyzed the mouse round spermatid UniGene library currently containing 2124 gene-oriented transcript clusters, predicting that 467 of them are testis-specific genes, and systematically identified 28 novel genes with evident testis-specific expression by in silico and in vitro approaches. We analyzed these genes by Northern blot hybridization and cDNA cloning, demonstrating the presence of additional transcript sequences in five genes and multiple transcript isoforms in six genes. Genomic analysis revealed lack of human orthologues for 10 genes, implying a relationship between these genes and male reproduction unique to mouse. We found that all of the novel genes are expressed in developmentally regulated and stage-specific patterns, suggesting that they are primary regulators of male germ cell development. Using computational bioinformatics tools, we found that 20 gene products are potentially involved in various processes during spermatogenesis or fertilization. Taken together, we predict that over 20% of the genes from the round spermatid library are testis-specific, have discovered the 28 authentic, novel genes with probable spermatogenic cell-specific expression by the integrative approach, and provide new and thorough information about the novel genes by various in vitro and in silico analyses. Thus, the study establishes on a comprehensive scale a new basis for studies to uncover molecular mechanisms underlying the reproductive process.