Hypoxia-inducible factor (HIF) is a heterodimer composed of HIF-alpha and -beta, and to date, three HIF-alpha subunits, HIF-1alpha, -2alpha, and -3alpha, have been identified. Among these HIF-alpha subunits, HIF-1alpha represses peroxisome proliferator activator gamma2 gene expression and then inhibits adipogenesis, and HIF-2alpha is induced during adipose differentiation and functions as a positive regulator of adipogenesis. We here found that like HIF-2alpha, HIF-3alpha was induced during 3T3-L1 adipose differentiation. Reporter gene analysis revealed that HIF-2alpha enhanced the promoter activity of the 5'-flanking region of the mouse HIF-3alpha gene (nucleotides -2710 to +56), while HIF-1alpha had no substantial effects on the promoter activity. These results suggested that HIF-2alpha, which was induced during adipogenesis, might regulate the HIF-3alpha gene expression. Furthermore, the 5'-deletion analysis revealed that the sequence between -251 and -228 in mouse HIF-3alpha promoter was essential in response to HIF-2alpha. We further examined the effect of ectopic expression of HIF-3alpha in 3T3-L1 cells on adipose differentiation and found that ectopic expression of HIF-3alpha at the early stage of differentiation induced the expression of several kinds of adipocytes-related genes and enhanced adipogenic potential. HIF-3alpha, which is induced by HIF-2alpha, might function as an accelerator of adipogenesis.