Calcitroic acid (1 alpha-hydroxy-23 carboxy-24,25,26,27-tetranorvitamin D(3)) is known to be the major water-soluble metabolite produced during the deactivation of 1 alpha,25-dihydroxyvitamin D(3). This deactivation process involves a series of oxidation reactions at C(24) and C(23) leading to side-chain cleavage and, ultimately, formation of the calcitroic acid. Like 1 alpha,25-dihydroxyvitamin D(3), 1 alpha,25-dihydroxyvitamin D(2) is also known to undergo side-chain oxidation; however, to date there has been no evidence suggesting that 1 alpha,25-dihydroxyvitamin D(2) undergoes side-chain cleavage. To investigate this possibility, we studied 1 alpha,25-dihydroxyvitamin D(2) metabolism in HPK1A-ras cells as well as the well characterized perfused rat kidney system. Lipid and aqueous-soluble metabolites were prepared for characterization. Aqueous-soluble metabolites were subjected to reverse-phase HPLC analysis. The major aqueous-soluble metabolite from both the kidney and cell incubations comigrated with authentic calcitroic acid on two reverse-phase HPLC columns of different chemistry. The putative calcitroic acid from the cell and kidney incubations was methylated and found to comigrate with methylated authentic standard on straight-phase and reverse-phase HPLC columns. The identity of the methylated metabolite from cell incubations was also confirmed by mass spectral analysis. These data show, for the first time, that calcitroic acid is a major terminal product for the deactivation of 1 alpha,25-dihydroxyvitamin D(2). Intermediates leading to the formation of the calcitroic acid in the 1 alpha,25-dihydroxyvitamin D(2) metabolism pathway are currently being studied.