The active form of vitamin D, 1,25-dihydroxvitamin D3 (1,25(OH)2D3), is a pleiotropic hormone whose actions include the regulation of calcium homeostasis, control of bone cell differentiation and modification of immune responses. Synthesis of 1,25(OH)2D3 from the major circulating metabolite, 25-hydroxyvitamin D3 (25(OH)D3), is catalysed by a mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D-1α-hydroxylase (1α-OHase). Although 1α-OHase is expressed predominantly in the kidney, extra-renal production of 1,25(OH)2D3 has also been demonstrated in tissues such as lymph nodes and skin. The tight regulation of 1α-OHase which occurs in both renal and peripheral tissues has made studies of the expression and regulation of this enzyme remarkably difficult. However, the recent cloning of mouse, rat and human cDNAs for 1α-OHase (CYP1α/Cyp1α) has enabled a more thorough characterization of this enzyme. In particular, analysis of the CYP1α gene has identified mutations causing the inherited disorder vitamin D-dependent rickets type 1, also known as pseudo-vitamin D deficiency rickets. Studies from our own group have focused on the distribution of 1α-OHase in both renal and extra-renal tissues. Data indicate that the enzyme is expressed throughout the nephron, suggesting discrete endocrine and paracrine/autocrine functions. Further immunohistochemical analyses have shown that the enzyme is widely distributed in extra-renal tissues, and this appears to be due to the same gene product as the kidney. Collectively, these observations have raised important new questions concerning the role of 1α-OHase in vitamin D signalling at a local level. The relationship between expression of protein for 1α-OHase and enzyme activity has yet to be fully characterized and may be dependent on membrane proteins such as megalin. Similarly, elucidation of the mechanisms involved in differential regulation of renal and extra-renal 1,25(OH)2D3 production will be essential to our understanding of the tissue-specific functions of 1α-OHase. These and other issues are discussed in the current review.