L-lysine, a cationic essential amino acid, has been reported to affect calcium transport in both intestine and kidney. In order to investigate whether this effect is associated with changes in cytosolic calcium homeostasis, we studied the effect of L-lysine deprivation on intracellular calcium concentration ([Ca2+]i), as well as 45Ca efflux and accumulation in normal human fibroblasts. Steady state [Ca2+]i, measured using fura-2 fluorescence in cells cultured for 18 hours in a L-lysine-free medium, was significantly higher than in cells grown in the presence of as little as 4 microM L-lysine. L-lysine deprivation also led to a significant decrease of 45Ca fractional efflux compared with cells grown in complete medium. This effect was paralleled by a significant decrease in 45Ca accumulation. Lack of L-arginine from the growth medium for the same time period had no effect on either [Ca2+]i, 45Ca efflux, or 45Ca accumulation rate. Presumably, the lack of L-lysine for a significant amount of time impairs the active mechanisms of calcium extrusion, which is only partially compensated by a reduction of calcium accumulation rate. This leads to an increased steady state [Ca2+]i. It is concluded that L-lysine is an important modulator of cytosolic calcium homeostasis.