The highly conserved 14-3-3 family of proteins, originally reported as brain-specific and then found in various somatic cells and oocytes, interacts with several important signal transduction kinases so that actually the 14-3-3 protein are considered as modulators of multiple signal transduction pathways. Here we show that a 14-3-3 protein is also expressed in the male germ cells, thus extending the protein cellular distribution to a cell line never reported to express 14-3-3 proteins. Screening of a mouse spermatogenic cells lambda gt11 cDNA library with affinity-purified polyclonal antibodies to the tyrosine kinase SP42 allowed the isolation of several positive clones. Sequencing of a positive cDNA clone revealed a 735-nucleotide open reading frame encoding a protein of 245 amino acids (27,778 Da). The predicted protein was found to be identical to the most recently discovered 14-3-3 isoform, the theta subtype from a rat brain. Here we demonstrate that 14-3-3 theta mRNA is highly expressed in testis and brain only. Western immunoblot analyses confirm the Northern blot data. Developmental Northern and Western blot analyses are consistent with an expression and translation of the 14-3-3 theta gene throughout spermatogenesis. However, analysis of RNA from purified populations of spermatogenic cells at different developmental stages and immunohistochemistry on adult testis sections reveal that within the testis the 14-3-3 theta gene products are most abundant in meiotic prophase spermatocytes, and, above all, in differentiating spermatids. Both testicular and epididymal spermatozoa are negative. The present study is the first report on the presence and molecular characterization of the 14-3-3 theta gene product in the male germ line. Our observations suggest that this specific member of the 14-3-3 protein family could play distinct modulatory roles in the complex development of the mammalian male germ cell lineage.