The efflux of radiolabelled organic cations from the tubular lumen into proximal tubular cells was investigated by using the stop-flow microperfusion method. The efflux rate increased in the sequence: N1-methylnicotinamide (NMeN+) < cimetidine < tetraethylammonium (TEA+) < N-methyl-4-phenylpyridinium (MPP+). Preloading the animals by i.v. infusion or pre perfusion of the peritubular capillaries with NMeN+ increased the efflux rate of MPP+. Luminal efflux was also augmented when the tubular solution was made alkaline with HCO3- or phosphate, whereby HCO3- is more effective than phosphate. Replacement of Na+ by Cs+ showed no effect. With i.v. preloading the animals with NMeN+ and with 25 mM HCO3- in the luminal perfusate the 2-s efflux follows kinetics with a Michaelis constant Km = 0.21 mmol/l and maximal flux Jmax = 0.42 pmol.cm-1.s-1 and a permeability term with P = 37.7 microns2.s-1. Comparing the apparent luminal inhibitory constant values for MPP+ (Kil,MPP+) with the apparent contraluminal Kicl,NMeN+ values of substrates of homologous series, it was found that (1) limitation by molecular size occurs at the contraluminal cell side earlier than at the luminal cell side; (2) affinity increases with hydrophobicity of the substrates at the luminal cell side, with a steeper or equal slope than at the contraluminal cell side; (3) affinity increases with basicity (i.e. pKa values) at the luminal cell side with a steeper slope than at the contraluminal cell side. Taken together, substrates with low hydrophobicity and low basicity interact at the luminal cell side more weakly than at the contraluminal cell side. On the other hand large, hydrophobic substrates have, at the luminal cell side, a higher affinity than at the contraluminal cell side. Many substrates, however, have equal affinity at the luminal and contraluminal cell sides.