Transport of long-chain fatty acids (LCFA) was studied in isolated rat renal cortical membrane vesicles to determine the mechanism of entry of these metabolic substrates into renal cells. Total uptake of palmitate into basolateral (BLMV) and brush-border membrane vesicles (BBMV) was determined with the use of a rapid filtration technique. In the presence of an inwardly directed H+ gradient (pHo = 6.0, pHi = 7.4) palmitate (0.26 microM) uptake went through a maximum ("overshoot") in BLMV but not in BBMV. The amount of palmitate nonspecifically bound to vesicles was subtracted from total uptake to determine intravesicular content. Peak values for intravesicular palmitate content were 26 +/- 9 pmol/mg protein, 2.5-fold higher than 60-min values of 10 +/- 2 pmol/mg (P less than 0.05, n = 6). The overshoot could not be accounted for by changes in voltage. An inwardly directed Na+ gradient was without effect on palmitate uptake in BLMV and BBMV. pH gradient-stimulated palmitate transport (15 s) in BLMV was subject to cis-inhibition by 4,4'-diisothiocyanatostilbene-,,2-disulfonic acid. Probenecid and p-aminohippurate efflux from BLMV resulted in counter-flow accumulation of palmitate. It is concluded that at least a portion of LCFA transport across the basolateral membrane is mediated and may occur via anion exchange.