Previous work suggested a relationship between glycine metabolism and the effect of L-asparaginase upon tumor cells. Therefore, L5178Y (sensitive) or L5178Y/L-ASE (resistant) ascites lymphoma cells were incubated with 14C-labeled glyoxylate, glycine, serine, or asparagine, and the metabolism to other amino acids was measured by high performance liquid chromatography. Metabolic differences between the two cells lines were found. Under control conditions, the interconversion rate of glycine and serine via serine hydroxymethyltransferase (SHMT) was higher in sensitive than in resistant cells. The transformation rate of glyoxylate to serine was also higher in sensitive cells. These results may indicate a difference in the activity of SHMT. An alternate explanation would be that transport or diffusion of serine and glycine into sensitive cells is greater than into resistant cells. Several crucial metabolic differences were observed between the two cell types when L-asparaginase was added. A key difference is the decrease of glycine synthesis from glyoxylate observed in the sensitive cells compared to resistant cells which show no change. This suggests that asparagine is used for transamination of glyoxylate. Also, only sensitive cells appear to compensate for L-asparaginase-induced loss of glycine formation from glyoxylate by increasing glycine synthesis from serine. Alterations in sensitive tumor glycine metabolism may be an important function of L-asparaginase anticancer activity.