The recently identified transport proteins organic cation transporter 1 (OCT1), OCT2, and extraneuronal monoamine transporter (EMT) accept dopamine, noradrenaline, adrenaline, and 5-hydroxytryptamine as substrates and hence qualify as non-neuronal monoamine transporters. In the present study, selective transport substrates were identified that allow, by analogy to receptor agonists, functional discrimination of these transporters. To contrast efficiency of solute transport, stably transfected 293 cell lines, each expressing a single transporter, were examined side by side in uptake experiments with radiolabeled substrates. Normalized uptake rates indicate that tetraethylammonium, with a rate of about 0.5 relative to 1-methyl-4-phenylpyridinium (MPP+), is a good substrate for OCT1 and OCT2. It was not, however, accepted as substrate by EMT. Choline was transported exclusively by OCT1, with a rate of about 0.5 relative to MPP+. Histamine was a good substrate with a rate of about 0.6 relative to MPP+ for OCT2 and EMT, but was not transported by OCT1. Guanidine was an excellent substrate for OCT2, with a rate as high as that of MPP+. Transport of guanidine by OCT1 was low, and transport by EMT was negligible. With the guanidine derivatives cimetidine and creatinine, a pattern strikingly similar to guanidine was observed. Collectively, these substrates reveal key differences in solute recognition and turnover and thus challenge the concept of "polyspecific" organic cation transporters. In addition, our data, when compared with previous studies, suggest that OCT2 corresponds to the organic cation/H+ antiport mechanism in renal brush-border membrane vesicles, and that EMT corresponds to the guanidine/H+ antiport mechanism in membrane vesicles from placenta and intestine.