Arginine transport in suspension-cultured cells of Nicotiana tabacum L. cv. Wisconsin-38 was investigated. Cells that were preincubated in the presence of Ca(2+) for 6 h prior to transport exhibited stimulated transport rates. After the preincubation treatment, initial rates of uptake were constant for at least 45 min. Arginine accumulated in the cells against a concentration gradient; this accumulation was not the result of exchange diffusion. Arginine uptake over a concentration range of 2.5 μM to 1 mM was characterized by simple Michaelis-Menten kinetics with a Km of 0.1 mM and a Vmax of 9,000 nmol g(-1) fresh weight h(-1). Transport was inhibited by several compounds including carbonylcyanide-m-chlorophenylhydrazone, 2,4-dinitrophenol, N,N'-dicyclohexylcarbodiimide, and N-ethylmaleimide. Inhibition by these compounds was not the result of increased efflux resulting from membrane damage. A variety of amino acids and analogs, with the exception of D-arginine, inhibited transport, indicating that arginine transport was mediated by a general L-aminoacid permease. Competition experiments indicated that arginine and lysine exhibited cross-competition for transport, with Ki values similar to respective Km values. Arginine transport and low-affinity lysine transport are probably mediated by the same system in these cells.