The present study examined the nature of the apical inward and outward L-3,4-dihydroxyphenylalanine (L-dopa) transporters in LLC-PK₁ cells and whether protein kinases differentially modulate the activities of these transporters. The apical inward transfer of L-dopa was promoted through an energy-dependent and sodium-insensitive transporter (Michaelis constant = 38 µM; maximum velocity = 2608 pmol · mg protein¹ · 6 min¹). This transporter was insensitive to N-(methylamino)-isobutyric acid but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC; IC₅₀ = 251 µM). Modulators of protein kinase A (cAMP, forskolin, IBMX, and cholera toxin), protein kinase G (cGMP, zaprinast, LY-83583 and sodium nitroprusside), and protein kinase C (phorbol 12,13-dibutirate and chelerythrine) failed to affect the accumulation of L-dopa. The Ca²⁺/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-dopa uptake (IC₅₀ of 72 and 55 µM, respectively). The inhibitory effect of calmidazolium on the accumulation of L-dopa was of the noncompetitive type. The organic anion inhibitor DIDS, but not p-aminohippurate, and the protein tyrosine kinase (PTK) inhibitor genistein significantly increased L-dopa accumulation, which was mainly due to inhibition of apical outward transfer of L-dopa. It is concluded that LLC-PK₁ cells take up L-dopa over the apical cell border through the L-type amino acid transporter, which appears to be under the control of Ca²⁺-calmodulin-mediated pathways. The apical outward transfer of L-dopa may be promoted through a DIDS-sensitive transport mechanism and appears to be under the tonic control of PTK.