The alpha-2 agonist clonidine is frequently used in transdermal therapeutic systems for antihypertensive therapy. This study was performed to characterize transport of clonidine into human keratinocytes. The uptake of [3H]clonidine was measured into monolayers of the human cell line HaCaT and normal human epidermal keratinocytes in primary culture. The uptake of clonidine was linear for up to 1min, independent of Na(+), but pH-dependent. Uptake was carrier-mediated with an affinity constant (K(t)) of 0.30mM and a maximal velocity (V(max)) of 15.7nmol/min per mg of protein. Diphenhydramine, guanabenz, procainamide, tryptamine, quinine, and quinidine, but not choline markedly inhibited clonidine uptake. We conclude that clonidine is transported into keratinocytes in a pH-dependent manner by a saturable uptake system different from the keratinocyte choline transporter. The substrate specificity of the system corresponds to that of the recently characterized system for tertiary amines. After diffusion of the drugs through the stratum corneum, this transport system might contribute to the passage of clonidine and diphenhydramine across the living epidermis after dermal administration.