The effect of substituted benzoylglycines (hippurates) and phenylacetylglycines on the transport of p-aminohippurate (PAH) was studied in basolateral (BLMV) and brush border membrane vesicles (BBMV) isolated from dog kidney cortex. The probenecid-sensitive part of 100 microM [3H]PAH uptake into BLMV and BBMV was measured in the presence and absence of 5 mM glycine conjugate. The benzoyl- and phenylacetylglycines studied were substituted in the 2-, 3-, or 4-position with an H, CH3, OCH3 or OH group. Benzoylglycines were stronger inhibitors of PAH transport than phenylacetylglycines and the inhibitory potency of the conjugates was in general lower against the transporter in BBMV than BLMV. The specificities of the transporters in both membranes appear to be very similar. The inhibitory potency of the benzoylglycines, expressed as the apparent inhibition constant (logKi), did not show a linear relationship with their lipophilicity as determined by reversed phase HPLC. Deviation from linearity was caused mainly by the 3-OH and 4-OH analogs, which showed a greater inhibitory potency than expected from their lipophilicity. Phenylacetylglycines only showed a small variation in logKi values, indicating that insertion of a CH2 group between the ring and the carbonyl practically abolishes the influence of the ring and its substituents. In conclusion, both hydrophobic and electronic properties are important determinants of affinity for the PAH transport system. An additional partially negative hydroxyl group in the ring, located preferably at the 3- or 4-position, increases the interaction with the transport system.