Proline uptake and metabolism has been examined in newborn Sprague-Dawley rat kidney and compared to that in adult animals. [14C]-Proline uptake by renal tubule fragments from newborn rats occurs at the same initial rate as in adult tubules, but at physiologic concentrations achieves significantly higher intracellular levels after 15 min of incubation. Considerable metabolism of the proline taken up was observed in tissue of both ages. Analysis of acid soluble and insoluble tubule fractions from newborn and adults indicates similar degrees of proline incorporation into protein and oxidation to CO2 relative to the amount of radioactivity taken up. A major difference exists, however, with respect to the labeled components of the acid extract: adult tubules convert [14C]-proline to metabolites at a rate twice that of newborn. Analysis of concentration-dependent uptake data reveals two distinct entry systems for proline in both isolated newborn and adult tubules. No difference in the Km or Vmax was found between the young and mature tubules.