The promoter/regulatory sequences responsible for the transcription of the rat inhibin α 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 α subunit gene. The CAT activity was elevated when the 2·0 kb 5′ flanking α subunit gene fragment was progressively shortened from its 5′ end, and a maximal increase was reached when the CAT gene was driven by an α subunit gene promoter extending to −163 bp. This construct was termed AαBstCAT. Furthermore, when either the −2·0 to −1·6 kb or the −2·0 to −1·0 kb α subunit DNA fragment was fused to AαBstCAT, the 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 α subunit gene, which was dependent upon the putative cAMP response element within the 67 bp α 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 α 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 α subunit gene in testicular MA-10 cells may, at least in part, be controlled by the upstream silencer(s) and NRE(s).