Previously, two promoters were identified for the rabbit FMO1 gene: a major, upstream promoter (P0) that initiates transcription from exon 0 and a second, minor promoter (P1) located approximately 200 bp downstream and initiating transcription from exon 1. Transcription initiation from the P0 promoter results in elimination of the exon 1 leader sequence from the mature transcript. In this report, we further define the major promoter and identify several positive and negative upstream regulatory domains employing deletion analysis and transient expression in HepG2 cells. Of interest, P0 and P1 were equally active in these assays. A 49-bp fragment spanning position -41 to +8 was found essential for the activity of P0 and also capable of basal transcriptional activity. Interestingly, this same 49-bp region was found necessary for P1 activity. Upstream of P0, three positive regulatory regions (positions -348 to -176, -757 to -584, and -1196 to -829) and two negative regulatory regions (positions -2120 to -1724 and 829 to -757) were identified using deletion mutants. Both P0 and P1 share the most proximate, positive regulatory domain but were regulated differentially by more distal 5' sequences. In addition to the upstream regulatory sequences, a potent negatively acting element was observed within intron 1. Using DNA fragments representing the most potent positive (position -348 to -176) and negative (position -829 to -757) regulatory sequences as probes, we demonstrate the formation of multiple specific DNA/protein complexes with protein factor(s) present in HepG2 nuclear extract.