Cytosolic purine nucleoside phosphorylase (PNPase) is a well known, and described enzyme which exists in a variety of organisms, both procaryotic and eucaryotic. More recently this enzyme was found in bovine liver mitochondria. The mitochondrial purine nucleoside phosphorylase was purified 63 fold and has a molecular weight of 48-60 kD. From Lineweaver-Burk plots apparent KM's of 23 microM for inosine, 42 microM for deoxyinosine, 40 microM for phosphate, 2 microM for hypoxanthine, and 163 microM for ribose-1-phosphate were calculated. Both 8-aminoguanosine (Ki = 0.5 microM) and araG (Ki = 381 microM) are inhibitors of the enzyme. The protein's isoelectric point (pI) was calculated at a pH of 4.2. Preliminary immunological work showed no cross-reactivity between epitopes on the mitochondrial protein and those on PNPase from human erythrocytes. The apparent KM's calculated for the mitochondrial enzyme are, with the exception of that using hypoxanthine, within the range commonly associated with KM's from the cytosolic species. The mitochondrial enzyme's molecular weight and pI are less than normally described. The enzyme's isolation from mitochondria, together with several unique characteristics, suggest that it is a separate protein from that found in the cytosol.