Reportedly, the initiating event in the renal uptake of gentamicin is its binding to anionic, plasma membrane phospholipids. Because parathyroid hormone is known to affect phospholipid metabolism, the plasma membrane binding and tissue accumulation of gentamicin were examined as a function of the parathyroid hormone status of the animal. The experiments were conducted by evaluating the parameters in isolated brush border and basolateral membranes from control, hyper- and hypoparathyroid rats. Scatchard analysis revealed that [125I]gentamicin bound with equal affinity to either membrane to a single class of noninteracting sites. The basolateral membrane had more binding sites than did the brush border, 28 +/- 0.5 vs. 1.8 +/- 0.3 nmol/mg of protein, respectively. Neither the affinity constants nor the number of binding sites were affected by the parathyroid hormone status of the donor animal. On the other hand, in the hypoparathyroid state the amount and the rate of gentamicin accumulation were less than in the hyperparathyroid state. The difference in accumulation cannot be explained on the basis of a change in the number or affinity of the putative receptor. Therefore, the alteration must reflect some difference subsequent to the binding site.