The janus locus of Drosophila melanogaster displays a very unusual organization. It comprises two partially overlapping genes, janA and janB, which are transcribed in the same orientation; the start of transcription of janB, the downstream gene, is located in the 3' exonic region of janA. Both genes are expressed during spermatogenesis. Transcription of janB is restricted to this developmental process, whereas janA is ubiquitously transcribed in both the somatic and germinal tissues of males and females. In order to delimit the cis-acting sequences regulating the transcription of janB, the expression of four chimeric janB-lacZ genes was examined in transgenic lines by Northern blot analysis, in situ hybridization and in situ histochemical staining for beta-galactosidase activity. Results showed that the testis-specific expression of the janB gene is mediated by a short DNA sequence (positions -174 to +107) which is located entirely within the last exon of the upstream janA gene. The tissue specificity of the expression of the janB gene is maintained when most of the janA coding and upstream sequences are deleted. Yet the presence in cis of an active janA gene leads to reduced accumulation of the janB-lacZ hybrid mRNA. This supports the hypothesis that janA transcription interferes with the function of the janB cis-regulatory elements. Our results also demonstrate that the 5' untranslated leader of the janB mRNA contains translational cis-acting elements, which completely block the translation of the janB-lacZ transcripts during the premeiotic stages of sperm development. A janB-lacZ construct was used to examine the sexual phenotype of the germline cells of masculinized XX transformer-2 (tra-2) flies. This has enabled us to confirm at the molecular level previous observations that the germline cells of these flies can enter the spermatogenic pathway of differentiation.