A steady-state level of about 240 nmol/mg (dry wt) occurs during lysine transport in Saccharomyces cerevisiae. No subsequent efflux of the accumulated amino acid was detected. Two transport systems mediate lysine transport, a high-affinity, lysine-specific system and an arginine-lysine system for which lysine exhibits a lower affinity. Preloading with lysine, arginine, glutamic acid, or aspartic acid inhibited lysine transport activity; preloading with glutamine, glycine, methionine, phenylalanine, or valine had little effect; however, preloading with histidine stimulated lysine transport activity. These preloading effects correlated with fluctuations in the intracellular lysine and/or arginine pool: lysine transport activity was inhibited when increases in the lysine and/or arginine pool occurred and was stimulated when decreases in the lysine and/or arginine pool occurred. After addition of lysine to a growing culture, lysine transport activity was inhibited more than threefold in one-third of the doubling time of the culture. These results indicate that the lysine-specific and arginine-lysine transport systems are regulated by feedback inhibition that may be mediated by intracellular lysine and arginine.