Evidence is presented for the existence of a new C-23 oxidation pathway for the metabolism of the hormonally active form of vitamin D3, namely 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). Homogenates of intestinal mucosa or kidney, but not liver, from rats and chicks convert 1,25(OH)2[26,27-3H]D3 into two new metabolites, 1 alpha,25-dihydroxy-23-oxo-vitamin D3 (1,25(OH)2-23-oxo-D3) and 1 alpha,25,26-trihydroxy-23-oxo-vitamin D3 (1,25,26(OH)3-23-oxo-D3), and an unknown metabolite(s) which has been possibly subjected to side chain modification/cleavage so that the tritium of the substrate has been converted into a form which is volatile. The isolation and chemical characterization of 1,25(OH)2-23-oxo-D3 and 1,25,26(OH)3-23-oxo-D3 from homogenates of chick intestinal mucosa has recently been described (Ohnuma, N., Kruse, J., Popjak, G., and Norman, A. W. (1982) J. Biol. Chem. 257, 5097-5102). Based on kinetic studies with chick intestinal homogenates, the proposed C-23 oxidation pathway is: 1,25(OH)2D3 leads to 1,25(OH)2-23-oxo-D3 leads to 1,25,26(OH)3-23-oxo-D3 leads to unknown metabolite with altered side chain. The relative enzyme activities of the C-23 pathway in tissues from vitamin D-replete chicks are: intestine, kidney liver, 18:3:less than 1. The activity of the C-23 pathway in homogenates of chick intestinal mucosa can be enhanced 10 x by prior priming of the birds with a single intravenous dose of 500 ng (1.3 nmol) of 1,25(OH)2D3; the induction of the enzyme activity is maximal by 3-6 h and returns to basal levels by 12 h. A comparison was made in chick and rat intestinal homogenates of this C-23 pathway and the previously known C-24 oxidation pathway, which converts 1,25(OH)2D3 into 1,24,25-trihydroxyvitamin D3 (1,24,25(OH)3D3); it was calculated that under these conditions 72% of the 1,25(OH)2D3 was metabolized by the C-23 oxidation pathway, 13% by the C-24 pathway, and only 14% by other as yet unspecified pathways. It is proposed that the newly discovered C-23 pathway for metabolism of 1,25(OH)2D3 by the target intestinal mucosa and kidney may play a prominent role under physiological conditions of controlling the tissue levels of this hormonally active form of vitamin D3.