Flavin-containing monooxygenases (FMOs) are important oxidative drug metabolizing enzymes. FMO3 is the primary human adult liver FMO enzyme, but is developmentally regulated. FMO3 promoter characterization using in vitro DNA binding assays with HepG2 cell and fetal and adult liver nuclear protein, as well as FMO3/reporter construct transient expression in HepG2 cells, provided evidence for specific mechanisms contributing to both developmental and constitutive adult regulation. NFY, USF1, an unidentified GC box binding protein, and YY1 appear to play major roles regulating constitutive FMO3 transcription, while Pbx(2) as a heterodimer with an unidentified Hox isoform also may contribute to FMO3 developmental expression.