Ornithine decarboxylase (ODC; EC 220.127.116.11) is an important enzyme in the synthesis of polyamines and is associated with growth and differentiation. Insulin stimulation of cardiac ODC has been proposed as a marker of the functional completion of the sympathetic pathway to the rat heart. However, earlier studies, using subsaturating substrate concentrations and a single time point measurement after insulin treatment, have been inconsistent concerning the postnatal age at which significant insulin stimulation of ODC occurs. The present study, using a validated near-saturating substrate assay, examines more thoroughly early neonatal insulin induction of cardiac ODC with respect to both the magnitude and the time course of response. Insulin (20 IU/kg s.c.) significantly increased ODC activity at several time points at each postnatal age measured (days 2, 5, 8, 15 and 22), with maximum ODC activity occurring by 2.5 to 3 hr after insulin injection at all ages. Insulin-stimulated ODC activity was increased over control levels by 86, 84, 87, 150 and 127% on days 2, 5, 8, 15 and 22, respectively. These results demonstrate that age is not a variable in the time of peak insulin stimulation of ODC activity and, in contrast to earlier reports, show that significant insulin induction of cardiac ODC activity occurs reliably across ages in the early postnatal period. The inconsistency of earlier studies may be due to a number of factors, including the use of subsaturating enzyme assays only, known to be subject to several types of error.