The common substrate for glutathione S-transferases (GSTs), 1-chloro-2,4-dinitrobenzene (CDNB), is an inhibitor of Escherichia coli growth. This growth inhibition by CDNB is enhanced when E. coli expresses a functional GST. Cells under growth inhibition have reduced intracellular GSH levels and form filaments when they resume growth. Based on this differential growth inhibition by CDNB we have developed a simple procedure to select for null-mutants of a human GST in E. coli. Null mutations in the human GST gene from hydroxylamine mutagenesis or oligonucleotide-directed mutagenesis can be selected for on agar plates containing CDNB after transformation. The molecular nature of each mutation can be identified by DNA sequence analysis of the mutant GST gene. We have identified three essential amino acid residues in an alpha class human GST at Glu31, Glu96, and Gly97. Single substitution at each of these residues, E31K, E96K, G97D, resulted in mutant GST proteins with loss of CDNB conjugation activity and failure in binding to the S-hexyl GSH affinity matrix. In contrast, a mutant GST (Y8F) resulting from substitution of the conserved tyrosine near the N terminus has much reduced CDNB conjugation activity but was still capable of binding to the S-hexyl GSH-agarose. Additional mutant GSTs with substitutions at position 96 (E96F, E96Y) and 97 (G97P, G97T, G97S) resulted in changes in both Km and kcat to different extents. The in vitro CDNB conjugation activity of the purified mutant enzymes correlate negatively with the plating efficiencies of strains encoding them in the presence of CDNB. Based on the x-ray structure model of human GST 1-1, two of these residues are involved in salt bridges (Arg19-Glu31, Arg68-Glu96) and the third Gly97 is in the middle of the helix alpha 4. Our results provide evidence in vivo that Tyr8, Gly97, and the two salt bridges are important for GST structure-function. This molecular genetic approach for the identification of essential amino acids in GSTs should be applicable to any GSTs with CDNB conjugation activity. It should also complement the x-ray crystallographic approach in understanding the structure and function of GSTs.