Steroid 21-hydroxylase is expressed at high levels in the adrenal gland, where it is required for the synthesis of mineralocorticoids and glucocorticoids. The cis-acting nucleotide sequences required for expression of murine 21-hydroxylase were assessed by stable transfection of Y1 adrenocortical tumor cells with 21-hydroxylase genes containing progressive deletions of the 5' flanking sequences. Expression of authentic 21-hydroxylase transcripts was quantitated by S1 nuclease protection assays using total RNA from pools of transfectant colonies. Removal of sequences from 1100 to 230 base pairs preceding the transcription initiation site did not significantly affect 21-hydroxylase transcription. However, an important regulatory element falls between 230 and 180 base pairs upstream of the transcription initiation site. Comparisons of the murine 5' flanking sequence with those of the human and bovine 21-hydroxylase genes revealed a 40-base pair region that is highly conserved among all three species. The conserved sequence in the murine gene occurs between 210 and 170 nucleotides 5' of the transcription initiation site; similar locations are seen in the human and bovine genes. These results thus define an important regulatory region which is required for expression of 21-hydroxylase in Y1 adrenal cells.