A temperature-sensitive Escherichia coli mutant defective for the ability to utilize L-asparagine as a sole nitrogen source was isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis.The mutation (mu) produces two distinct phenotypic effects. (1) Mutant strains grow poorly at high temperature on minimal plates containing asparagine as the sole nitrogen source; this effect is greatly exacerbated by the presence of methionine. (2) Mutant strains utilize Lasparagine as a nitrogen source three to four times more efficiently at permissive temperatures than the wild-type strains. The mutation maps at 32.4 min on the E. coli chromosome, within the E. coli cotransduction gap. Mutant strains produce normal amounts of thermo-stable Lasparaginase I activity. The mutation therefore affects a component of the asparagine utilization system other than the catabolism of asparagine within the cell; it probably affects asparagine uptake.