1. Rat liver mitochondrial matrix peptidyl-prolyl cis-trans-isomerase (PPIase) has been purified. The major form of the enzyme has a molecular mass of 18.6 kDa, with a minor active component of 17.6 kDa. 2. The second-order rate constant for cyclosporin A binding to the enzyme was determined from the time-dependence of the inhibition of PPIase by low concentrations of cyclosporin A and found to be 0.9 microM-1.s-1 at 10 degrees C. 3. The Ki for cyclosporin A inhibition of the enzyme was 3.6 nM, and the half-life for dissociation of the enzyme-inhibitor complex was 3.6 min. 4. From the specific activity of the pure enzyme it can be calculated that isolated liver mitochondria contain approx. 45 pmol of enzyme per mg of total mitochondrial protein. Higher values estimated previously [Halestrap & Davidson (1990) Biochem. J. 268, 153-160] are explained by the use of a short (30 s) preincubation period of the enzyme with cyclosporin, which is insufficient to allow full equilibration of the binding of the inhibitor to the PPIase. 5. N-Terminal sequencing of the 18.6 and 17.5 kDa forms of PPIase show the presence of mitochondrial presequences of 13 and three amino acids respectively, with the remaining sequence having a strong sequence similarity to other cyclophilins. 6. Parallel purification and N-terminal sequencing of rat cytosolic PPIase showed the two proteins to have significant differences, implying that they are probably products of separate genes.