High vitamin D intake is associated with reduced insulin resistance. Expression of extra-renal 1alpha,25-dihydroxyvitamin D hydroxylase (1alpha-hydroxylase) has been reported in several tissues and contributes to local synthesis of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D) from the substrate 25-hydroxyvitamin D (25OHD). Expression and dietary regulation of 1alpha-hydroxylase in tissues associated with energy metabolism, including adipose tissue, has not been assessed. Male Wistar rats were fed a high calcium (1.5%) and high vitamin D (10,000IU/kg) or a low calcium (0.25%), low vitamin D (400IU/kg) with either a high fat (40% energy) or high sucrose (66% energy) dietary background for 14 weeks. Expression of 1alpha-hydroxylase, assessed by real time PCR, was detected in adipose tissue and did not differ with dietary level of calcium and vitamin D. 1alpha-Hydroxylase mRNA was also detected in 3T3-L1 preadipocytes and 25OHD treatment at 10nM levels induced 1,25(OH)(2)D responsive gene, CYP24, and this response was reduced in the presence of the p450 inhibitor, ketoconazole. In addition, (3)H 25OHD was converted to (3)H 1,25(OH)(2)D in intact 3T3-L1 preadipocytes. Cumulatively, these results demonstrate that 1alpha-hydroxylase is expressed in adipose tissue and is functional in cultured adipocytes. Thus, the capacity for local production may play a role in regulating adipocyte growth and metabolism.