We have studied the submitochondrial localization of guinea-pig kidney 25-hydroxycholecalciferol 1 alpha-hydroxylase. Treatment of the mitochondrial-enriched fraction with recrystallized digitonin produced mitoplasts bordered by a single membrane and with intact matrix. They contained nearly 90% of the 25-hydroxycholecalciferol 1 alpha-hydroxylase activity and nearly 100% of the cytochrome-c:oxygen oxidoreductase. Amine:oxygen oxidoreductase activity remained mainly in the outer membrane fraction. These data show that 25-hydroxycholecalciferol 1 alpha-hydroxylase has a distribution similar to that of steroid hydroxylases.