The promoter/regulatory sequences responsible for the transcription of the rat inhibin alpha subunit gene in the testis were identified by the transient expression in an MA-10 Leydig tumour cell line of a bacterial reporter gene, chloramphenicol acetyltransferase (CAT), which was driven by different regions of the 5' flanking sequence of the inhibin alpha subunit gene. The CAT activity was elevated when the 2.0 kb 5' flanking alpha subunit gene fragment was progressively shortened from its 5' end, and a maximal increase was reached when the CAT gene was driven by an alpha subunit gene promoter extending to -163 bp. This construct was termed A alpha BstCAT. Furthermore, when either the -2.0 to -1.6 kb or the -2.0 to -1.0 kb alpha subunit DNA fragment was fused to A alpha BstCAT, and CAT activity was markedly suppressed, indicating the presence of negative regulatory DNA elements (NREs) in the upstream region of the gene. The cyclic AMP (cAMP) responsiveness of the alpha subunit gene, which was dependent upon the putative cAMP response element within the 67 bp alpha subunit promoter, was not affected by the upstream NREs. The inhibitory effect was also demonstrated when the -2.0 to -1.0 kb fragment was placed in either orientation with respect to the alpha subunit promoter or to a thymidine kinase promoter, suggesting that the NRE(s) can act as a silencer. Based on our observations we conclude that the basal expression of the rat inhibin alpha subunit gene in testicular MA-10 cells may, at least in part, be controlled by the upstream silencer(s) and NRE(s).