3-Hydroxy-3-methylglutaryl (HMG)-CoA metabolism is compartmentalized in mitochondria, endoplasmic reticulum, and peroxisomes. We investigated the subcellular distribution of HMG-CoA lyase (HL), which is found principally in mitochondria but in which we observed the potential peroxisomal targeting motif cysteine-lysine/arginine-leucine at the carboxyl terminus. We used differential and density gradient centrifugation to separate peroxisomes and mitochondria in liver homogenates of outbred CD-1 mice. Peroxisomal fractions contained 6.4% of total HL activity in mouse liver and 5.6% in human liver. Liver peroxisomal HL activity increased 2.3-2.5 times following induction of peroxisomal proliferation by clofibrate administration. Western blotting with anti-human HL antibodies confirmed the presence of immunoreactive HL in peroxisomal fractions. Mouse liver peroxisomal HL is distinct from mitochondrial HL, measuring approximately 2.5 kDa more by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By fast protein liquid chromatofocusing analysis, the pI of peroxisomal HL is 7.3, in contrast to 6.2 for mitochondrial HL. These results are consistent with noncleavage of the mitochondrial leader peptide in peroxisomal HL. A distinct species of enzymatically active HL exists in peroxisomes and may play a role in HMG-CoA metabolism in that organelle.