We studied pre- and postnatal changes in total creatine kinase (CK) activity, mitochondrial creatine kinase (Mi-CK) activity and immunochemical reactivity with anti-Mi-CK antibodies in skeletal muscle specimens from 12 infants, 10 of them preterm born, after a pregnancy varying between 28 and 40 weeks. Our results demonstrate that Mi-CK is present in fetal human quadriceps muscle and that the specific activity of Mi-CK increases during prenatal development from week 28 to 40 by a factor about two. Generally, adult levels have not been reached at birth, indicating a further postnatal increase of the activity of the enzyme. The Mi-CK protein content also increases during prenatal development. These results suggest that in human skeletal muscle the expression and accumulation of Mi-CK starts at mid-gestation, later than is known to occur for cytosolic CK.