The in vitro relationship between nutritional factors, proliferative status of tumor cells, and the cytotoxic action of cytosine arabinoside (ara-C) was investigated. The reduction in the concentration of only one essential amino acid, L-isoleucine, in the growth medium of A(T1)C1-3 hamster fibrosarcoma cells decreased DNA synthesis in this cell population and slowed the rate of progression of G1 phase cells into S phase of the cell cycle. The complete omission of isoleucine from the growth medium blocked the progression of G1 phase cells into S phase and prevented the cytotoxic action of ara-C. The addition of isoleucine to the isoleucine-deprived cells permitted these cells to enter the S phase and restored their sensitivity to the cytotoxic action of ara-C. When G1 phase cells were placed in a medium containing reduced levels of all the amino acids and vitamins there was a prolongation of the G1 phase. Since medium with low levels of amino acids produced a delay in the entry of G1 phase cells into the S phase, the time interval in which these cells were most sensitive to the cytotoxic action of ara-C was different for G1 phase cells placed in medium with adequate levels of all the amino acids. These in vitro data indicate that nutritional factors can markedly effect the proliferation of tumor cells and the cytotoxic action of ara-C.