Abstract This study presents evidence for the first time that rat liver peroxisomes contain a hydroxylase capable of converting 3α, 7α, 12α,- trihydroxy-5β-cholestane to a cholestanetetrol. Furthermore, this hydroxylase differs from both the mitochondrial and microsomal enzymes in its response to various co-factors. Highly purified peroxisomal, mitochondrial, and microsomal fractions from cholestryamine-treated rats were incubated with [22(23)- 3H]3α, 7α, 12α, -trihydroxy-5β-cholestane under a variety of conditions. The products were acidified, extracted, and subjected to thin-layer chromatography to determine the amount of cholestanetetrol produced. The identification of the 25- and 26-hydroxylated products from the incubations with the microsomes was confirmed by gas chromatography-mass spectrometry. Peroxisomal fractions incubated with a NADPH-generating system, Mg 2+, and ATP showed a rate of 40 pmol/min/mg conversion of 3α, 7α,12α, -trihydroxy-5β-cholestane to a cholestanetetrol. Co-factor studies indicated that both the peroxisomal and mitochondrial hydroxylase activities were dependent on NADPH, Mg 2+, and ATP (with different concentration requirements) whereas the microsomal hydroxylase(s) required only NADPH. An abstract of this work has been published (1).