Para-aminohippurate (PAH) is secreted at different rates in S1, S2, and S3 segments of isolated perfused proximal tubules of rabbit kidney. To characterize PAH transport we determined the maximal rate of secretion (Vmax) and the apparent Michaelis constant (Km) for each segment by examining the relationship between bath concentration of PAH and net PAH secretion (Jb leads to lPAH) transposed for Lineweaver-Burk analysis. The passive component of secretion for all segments was estimated by slope analysis at relatively high concentrations of PAH, by the component of PAH secretion insensitive to inhibition by probenecid, and, additionally, in S2 segments, from PAH efflux from lumen to bath. Subtraction of the passive component from Jb leads to lPAH (probenecid method) gave Vmax values for S1, S2, and S3 segments of 1,097 +/- 336 (n = 6), 7,430 +/- 1,338 (n = 6), 1,647 +/- 138 (n = 8) X 10(-15) mol.min-1.mm-1 (+/- SE) and apparent Km values of 139 +/- 37 (n = 6), 195 +/- 37 (n = 6), and 113 +/- 16 (n = 6) X 10(-6) M, respectively. Thus, Vmax for S2 greater than S3 congruent to S1, whereas apparent Km was not consistently different among the segments. On the basis of these results we suggest that axial heterogeneity of PAH secretion may reflect an increased basolateral membrane density of PAH transporters of common affinity in the S2 segment of the proximal tubule.