<jats:title>ABSTRACT</jats:title> <jats:p> An <jats:italic>Escherichia coli</jats:italic> strain deficient in <jats:italic>p</jats:italic> -aminobenzoate synthesis was mutagenized, and derivatives were selected for growth on folic acid. Supplementation was shown to be due to <jats:italic>p</jats:italic> -aminobenzoyl-glutamate present as a breakdown product in commercial folic acid preparations. Two classes of mutations characterized by the minimum concentration of <jats:italic>p</jats:italic> -aminobenzoyl-glutamate that could support growth were obtained. Both classes of mutations were genetically and physically mapped to about 30 min on the <jats:italic>E. coli</jats:italic> chromosome. A cloned wild-type gene from this region, <jats:italic>abgT</jats:italic> (formerly <jats:italic>ydaH</jats:italic> ) could confer a similar <jats:italic>p</jats:italic> -aminobenzoyl-glutamate utilization phenotype on the parental strain. Interruption of <jats:italic>abgT</jats:italic> on the plasmid or on the chromosome of the mutant strain resulted in a loss of the phenotype. <jats:italic>abgT</jats:italic> was the third gene in an apparent operon containing <jats:italic>abgA</jats:italic> , <jats:italic>abgB</jats:italic> , <jats:italic>abgT</jats:italic> , and possibly <jats:italic>ogt</jats:italic> and might be regulated by a divergently transcribed LysR-type regulator encoded by <jats:italic>abgR</jats:italic> . Two different single-base-pair mutations that gave rise to the <jats:italic>p</jats:italic> -aminobenzoyl-glutamate utilization phenotype lay in the <jats:italic>abgR-abgA</jats:italic> intercistronic region and appeared to allow the expression of <jats:italic>abgT</jats:italic> . The second class of mutation was due to a tandem duplication of <jats:italic>abgB</jats:italic> and <jats:italic>abgT</jats:italic> fused to <jats:italic>fnr</jats:italic> . The <jats:italic>abgA</jats:italic> and <jats:italic>abgB</jats:italic> gene products were homologous to one another and to a family of aminoacyl aminohydrolases. <jats:italic>p</jats:italic> -Aminobenzoyl-glutamate hydrolysis could be detected in extracts from several of the mutant strains, but intact <jats:italic>abgA</jats:italic> and <jats:italic>abgB</jats:italic> were not essential for <jats:italic>p</jats:italic> -aminobenzoyl-glutamate utilization when <jats:italic>abgT</jats:italic> was supplied in <jats:italic>trans</jats:italic> .