The diazepam binding inhibitor [DBI, also known as acyl-CoA-binding protein, (ACBP), or endozepine] is a 10-kD protein that has been suggested to be involved in the regulation of several biological processes such as acyl-CoA metabolism, steroidogenesis, insulin secretion, and gamma-aminobutyric acid type A (GABA(A))/benzodiazepine receptor modulation. DBI has been cloned from vertebrates, insects, plants, and yeasts. In mammals, DBI is expressed in almost all the tissues studied. Nevertheless, DBI expression is restricted to specific cell types. Here we have studied DBI gene expression in the germ-line cells of rat testis. The DBI gene was intensively transcribed in postmeiotic round spermatids from stages VI to VIII of the seminiferous epithelial cycle. A prominent, spermatid-specific upstream transcription initiation site was identified in addition to the multiple common transcriptional initiation sites found in the somatic tissues. However, no DBI protein was detected in round spermatids, suggesting that the DBI transcripts were translationally arrested. The DBI protein was detected in the late spermatogenic stages starting from elongating spermatids from step 18 (stage VI) onward. The DBI protein was also detected in mature spermatozoa and in ejaculated human sperms. The majority of DBI was located at the middle piece of the spermatozoons tail enriched with mitochondria. On the basis of this observation and the well-established role of DBI in acyl-CoA metabolism, we propose that DBI expression in spermatozoa reflects the usage of fatty acids as a primary energy source by spermatozoa. The biological function of DBI in spermatozoa could thus be related to the motility function of sperm.