Abstract A rapid assay of the ability of intact cells (10–50 mg) to synthesize l -asparagine and l -aspartic acid, and its application to the evaluation of potential asparagine analogs has been devised. Washed cell preparations were incubated at 37° in Eagle's medium (supplemented with l -glutamine, 2 mM, and l -aspartic acid, 0.05 mM), and the amounts of l -asparagine and l -aspartic acid formed were determined with an enzymatic assay. Murine tumor cells resistant to l -asparaginase produced 1.0 to 1.6 nmoles l -asparagine/mg cells/hr, while sensitive cells produced less than 0.1 nmole/mg cells/hr. The newly synthesized l -aspartic acid was largely localized m the cells; the newly synthesized l -asparagine, however, was distributed both in the cells and in the medium. 5-Diazo-4-oxo- l -norvaline (0.5 mM) and 5-chloro-4-oxo- l -norvaline (0.05 mM) inhibited l -asparagine biosynthesis by P815Y ascites cells 88 and 95 per cent respectively; no significant inhibition of l -aspartic acid biosynthesis was observed with either analog. 5-Bromo-4-oxo- l -norvaline (0.1 mM) caused 78 per cent inhibition of l -asparagine biosynthesis, but also reduced the biosynthesis of l -aspartic acid to 52 per cent.