The secretory transport of tetraethylammonium (TEA) was investigated in perfused and nonperfused isolated S1, S2, and S3 segments of proximal tubules from rabbit kidneys. In the perfused tubules the transepithelial net secretory flux and in nonperfused tubules the TEA cellular uptake were saturable (Km = 67 microM, Vmax = 2,480 fmol X min-1 X mm-1 in perfused S2 segments), energy dependent, and inhibited by mepiperphenidol. The net secretory flux of TEA (J b leads to j TEA) at a bath TEA concentration of 40 microM differed for the three segments and decreased in the order S1 greater than S2 greater than S3. The concentration of TEA in the perfusate leaving the tubule was approximately twice as great and the intracellular TEA concentration approximately 40 times as great as that in the bath. In nonperfused segments (40 microM TEA in the incubation medium) the TEA tissue water-to-medium ratio reached 100. In the three segments the ability to accumulate TEA across the peritubular membrane, thus, was similar, but the transepithelial secretory flux differed significantly. The differences in secretory rate between the three segments presumably result from differences in the luminal membrane permeability.