Renal tubular reabsorption of 3H and 14C labelled L-proline was measured in vivo et situ by continuous microperfusion of single proximal tubules of the rat. The reabsorption is shown to be saturable. Passive diffusion plays a relatively small role in the reabsorption. A maximum possible permeability coefficient of 25 micrometers 2.s-1 for proline was calculated. Two transport systems were found, one with a small affinity and a high capacity, the other with a very high affinity and a small capacity. The following values were estimated. Jmax 1 = 2.6 +/- 0.28 (SEM) nmol.m-1.S-1 Km1 = 11.8 +/- 1.7 (SEM) mmol.1-1 Jmax 2 = 9.6 +/- 1.92 (SEM) pmol.m-1.s-1 Km2 = 29.3 +/- 7.8 (SEM) mumol.1-1. Whereas the first system reabsorbs the bulk of the filtered load, the activity of the second system explains the extremely small amount of proline found in the final urine. Diisopropylphosphorofluoridate--a specific inhibitor of dipeptidyl peptidase IV--decreases the reabsorption of L-proline and L-alanine but has no influence on the reabsorption of the basic amino acid L-arginine and the acidic amino acid L-glutamic acid. This result correlates with a recent speculation that dipeptidyl peptidase IV is involved in proline and alanine reabosrption.