The vitamin D metabolite, 24,25-dihydroxyvitamin D-3, is made from 25-hydroxyvitamin D-3 by an enzyme (25-hydroxyvitamin D-3 24-hydroxylase) located in the renal mitochondria of vitamin D and calcium-replete chicks. The apparent Km for 25-hydroxyvitamin D-3 for this reaction was found to be 3 x 10(-7) M, although simple Michaelis-Menten kinetics were not observed at the higher substrate concentrations. Enzymatic activity was reduced by the mitochondrial metabolic inhibitors, antimycin A and dinitrophenol, as well as the inhibitors of mixed function oxidases, 2,4-dichloro-6-phenylphenoxyethylamine, 2-diethylaminoethyl-2,2-diphenylvalerate and metyrapone. Increasing the calcium concentration from 0 to 1 x 10(-5) M or the potassium concentration from 0 to 50 mM stimulated enzymatic activity 4- to 8-fold. Increasing the phosphate or acetate concentration from 0 to 50 mM stimulated enzymatic activity 6-fold. Raising the CO2/HCO3(-) content from 0/0 to 10%/10 mM stimulated enzymatic activity 5-fold. The effects of the cations were additive to those of the anions except in the presence of 50 mM phosphate or acetate. The effect of ions on 24,25-hydroxyvitamin D-3 production by renal mitochondria from vitamin D-replete chicks resembles the effect of these ions on production of 1,25-dihydroxyvitamin D-3 by renal mitochondria from vitamin D-deficient chicks, suggesting that the same mechanisms may be utilized.