Active and specialized transmembrane transport systems are responsible for the functional inactivation of catecholamines. Uptake2, the classical extraneuronal uptake system, and rOCT1, a recently cloned organic cation transporter, share a number of properties. The present study was undertaken to investigate putative differences between these two transporters that might clarify their relative physiological roles. Uptake of [3H]MPP+ ([3H]1-methyl-4-phenylpyridinium) by Caki-1 cells (to study uptake2) and by primary cultured rat hepatocytes (to study rOCT1) was kinetically and pharmacologically characterized. In both cell types, [3H]MPP+ was avidly taken up and accumulated. All compounds tested (catecholamines, metanephrines and corticosterone) inhibited [3H]MPP+ uptake, albeit with different potencies. In Caki-1 cells, the ranking order of inhibitory potency was: (-)isoprenaline > (-)adrenaline >> (-)noradrenaline > dopamine. Metanephrine and normetanephrine were equipotent. Corticosterone had an IC50 of 102 nM. In cultured hepatocytes, the ranking order of inhibitory potency was: (-)isoprenaline > dopamine > (-)adrenaline >> (-)noradrenaline. Metanephrine was about seven times more potent than normetanephrine. Corticosterone had an IC50 of 72 microM, being about 700-fold less potent in inhibiting rOCT1 than uptake2. The results showed that uptake2 and rOCT1 can be clearly distinguished on a functional basis. On the one hand, uptake2 prefers adrenaline among the endogenous catecholamines, whereas rOCT1 has similar affinity for adrenaline and dopamine. On the other hand, corticosterone and normetanephrine are significantly more potent in inhibiting uptake2 than rOCT1. The results are compatible with a possible physiological role of corticosteroids in the modulation of adrenaline effects in tissues equipped with uptake2, without significant interference with the hepatic and renal excretion of catecholamines.