Glutathione S-transferases (GSTs, EC 2.5.1.18) are a family of multi-functional enzymes involved in biodegradation of several herbicide classes. The ability of the maize isoenzyme GST I to detoxify from the acetanilide herbicide alachlor was investigated by steady-state kinetics and site-directed mutagenesis studies. Steady-state kinetics fit well to a rapid equilibrium random sequential bi-bi mechanism with intrasubunit modulation between GSH binding site (G-site) and electrophile binding site (H-site). The rate-limiting step of the reaction is viscosity-dependent and thermodynamic data suggest that product release is rate-limiting. Three residues of GST I (Trp12, Phe35 and Ile118), which build up the xenobiotic binding site, were mutated and their functional and structural roles during alachlor conjugation were investigated. These residues are not conserved, hence may affect substrate specificity and/or product dissociation. The work showed that the key amino acid residue Phe35 modulates xenobiotic substrate binding and specificity, and participates in k(cat) regulation by affecting the rate-limiting step of the catalytic reaction. Trp12 and Ile118 do not seem to carry out such functions but instead, regulate the K(m) for alachlor by contributing to its productive orientation in the H-site. The results of the present work have practical significance since this may provide the basis for the rational design of new engineered GSTs with altered substrate specificity towards herbicides and may facilitate the design of new, more selective herbicides.