In order to locate the promoter region of the human alpha 2A adrenergic receptor gene we used RNase protection analysis and antisense RNA probes to map the cap site of the alpha 2 transcripts. Prior sequence analysis has shown two potential TATA box motifs in the human alpha 2A adrenergic receptor gene, TATATAT and TATAAAA, located 427 and 1037 base pairs (bp), respectively, upstream of the protein coding region. RNase protection experiments and primer extension show that transcription starts downstream of the distal TATAAAA, indicating that the 5'-untranslated region is approximately 1 kilobase in length. We have used the chloramphenicol acetyltransferase reporter gene and transient transfection into HT29, a human adenocarcinoma cell line that expresses the alpha 2A receptor, to show that as little as 150 bp upstream of the cap site can direct transcription. Sequence analysis shows that although this region contains the TATA box motif it lacks a CCAAT box motif. DNase I footprint analysis of a fragment from -17 to -193 (where +1 is the transcription initiation site), using nuclear extracts from HT29, showed hypersensitive sites (-68/-69) and two protected regions: -70 to -87, which includes a 10-bp palindrome, and -92 to -105, which includes a GC box, a common motif for Sp1 nuclear factor binding. Gel mobility shift assays indicate that Sp1 or a related factor may bind to this GC box. Deletion of the GC box and the palindrome from chloramphenicol acetyltransferase constructs abolishes transcription. We propose that these cis sequences may function in lieu of a CCAAT box to regulate transcription of the human alpha 2A adrenergic receptor gene.