After transport across several epithelial barriers including the blood-brain barrier, clonidine interacts with alpha(2)-adrenergic receptors and imidazoline binding sites in the brain. We hypothesized that neuronal cells take up clonidine thereby removing the drug from the extracellular fluid compartment. Uptake of [(3)H]clonidine into SH-SY5Y neuroblastoma cells was linear for up to 1 min, unaffected by inside directed Na(+) or Cl(-) gradients but strongly inhibited by an outside pH of 6.0. The cells accumulated [(3)H]clonidine 50-70-fold uphill against a concentration gradient. Unlabeled clonidine, guanabenz, imipramine, diphenhydramine, maprotiline, quinine and the endogenous monoamine phenylethylamine (2 mM) strongly inhibited the [(3)H]clonidine uptake by 60-95%. Tetraethylammonium, choline and N-methyl-4-phenylpyridinium had no effect. The accumulation at pH 7.5 was saturable with an apparent Michaelis-Menten constant (K(t)) of 0.7 mM. We conclude that SH-SY5Y cells not only bind clonidine to extracellular receptors but also take up the drug rapidly by a specific and concentrative mechanism.