We have sequenced 4.2 kb of the 5' flanking region of the human beta-glucuronidase gene, compared this sequence to the 5' upstream sequence reported for the murine gene, determined the transcription start sites of the human gene, and studied expression of human minigene deletion constructs in COS cells. The 200 bp immediately 5' to the translation initiation codon have a high G + C content (72%) and contain no TATA box, two properties commonly associated with "housekeeping genes." The sequence 5' to -200 bp contains seven Alu repetitive elements which account for more than 50% of this flanking sequence. From deletion analysis of minigene constructs, 200 bp of 5' sequence appeared sufficient for maximal expression in transfected COS cells. S1 nuclease protection analysis showed that transcription initiates from a cluster of sites around -30 bp in all tissues examined. In some cases, a low but detectable level of transcription also initiates 126 bp upstream of the ATG. Inspection of the sequence surrounding both start sites revealed some similarity to the recently described "initiator" transcriptional control element (S.T. Smale and D. Baltimore (1989), Cell 57: 103-113). Comparison of the 5'flanking sequence with that available from the murine beta-glucuronidase gene reveals only one 28-bp highly conserved region, which surrounds the -126 start site.