To understand the basolateral p-aminohippurate (PAH) transporter in the S3 segment of rabbit proximal tubules and its relationship to the transporter in the S2 segment, we measured the 30-s uptake and efflux of PAH across the basolateral membrane of single isolated S3 segments at 37 degrees C in bicarbonate-buffered media. Kinetic analysis of uptake data revealed a concentration of PAH at one-half Jmax of approximately 107 microM (same as in the S2 segment) but a Jmax of 600 fmol.min-1.nl-1 (one-tenth that of S2 segment). The coefficient for efflux across the basolateral membrane was also only one-sixth to one-tenth of that in the S2 segment. These data suggest that the basolateral PAH transporter is the same in both segments but that there are fewer transporters in the S3 than in the S2 segment. However, the apparent inhibitor constant values for cis-inhibition by probenecid (approximately 29 microM in S3, approximately 15 microM in S2) and by alpha-ketoglutarate (alpha-KG) in the presence of LiCl (approximately 40 microM in S3, approximately 160 microM in S2) suggest that the transporters may not be identical in the two segments. In bicarbonate-buffered medium, preloading the tubules with 100 microM alpha-KG did not trans-stimulate PAH uptake across the basolateral membrane, whereas preloading with 1.0 microM alpha-KG caused a significant stimulation of 43%. However, in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered medium, preloading the tubules with 100 microM alpha-KG caused a twofold increase in PAH uptake.(ABSTRACT TRUNCATED AT 250 WORDS)