L-Alanosine, an analogue of aspartic acid which inhibits the conversion of inosine monophosphate to adenosine monophosphate (AMP), was evaluated in L1210 cells as a modulator of 5-fluorouracil (FUra) and 5-fluorouridine (FUrd) metabolism. L-Alanosine resulted in increased intra-cellular levels of 5-phosphoribosyl-1-pyrophosphate (PRPP), enhanced FUra metabolism to ribonucleotide derivatives, and resulted in more FUra residues incorporated into RNA. Sequential addition of L-alanosine and FUra also resulted in synergistic cytotoxicity as determined by soft agar cloning. Adenine antagonized these biochemical and biological effects of L-alanosine. L-Alanosine also augmented the rate at which FUrd was metabolized and was also associated with a greater incorporation of the FUra residues into RNA. Cytotoxicity after sequential L-alanosine and FUrd was also synergistic. The mechanism by which L-alanosine altered the metabolism of FUrd, however, was different from the way in which it enhanced FUra metabolism in that aspartic acid and not adenine was able to reverse the effects of L-alanosine on FUrd metabolism and cytotoxicity. L-Alanosine appeared to augment the RNA-directed activity of FUra and FUrd in that there was no correlation between the enhanced metabolism and cytotoxicity of these two fluoropyrimidines and either levels of fluorodeoxyuridylate (FdUMP) which inhibits thymidylate (TMP) synthetase or inhibition of the ability of cells to incorporate deoxyuridylate into acid-precipitable material.