Alpha class glutathione S-transferase, isozyme A1-1, is a dimer (51 kDa) of identical subunits. Using the crystal structure, two main areas of subunit interaction were chosen for study: (1) the hydrophobic ball and socket comprised of Phe52 from one subunit fitting into a socket formed on the other subunit by Met94, Phe136, and Val139 and (2) the Arg/Glu region consisting of Arg69 and Glu97 from both subunits. We introduced substitutions of these residues, by site-directed mutagenesis, to evaluate the importance of each at the subunit interface and to determine if monomeric enzymes could be generated using single mutations. Mutating each residue of the socket region to alanine results in little change in the kinetic parameters, and all are dimeric enzymes. In contrast, when Phe52, the ball residue, is replaced with alanine, the enzyme has very low activity and a weight average molecular mass of 31.9 kDa, as determined by sedimentation equilibrium experiments. Substitutions for Glu97 which eliminate the charge cause no appreciable changes in the kinetic parameters or molecular mass. Eliminating the charge on Arg69 (as in R69Q) results in a dimeric enzyme; however, when the charge is reversed (as in R69E), the weight average molecular mass is greatly shifted toward that of the monomer (33 kDa) and the changes in kinetic parameters are reasonably small. We determined the molecular masses in the presence of glutathione for F52A and R69E to ascertain whether the monomeric species retains activity. For R69E, it appears that the monomer is active, albeit less so than the dimer, while for F52A, the monomer and dimer both appear to exhibit very low activity. The dimeric species is needed to obtain high specific activity. We conclude that, of the residues that were studied, Phe52 and Arg69 are the major determinants of dimer formation and a single mutation at either position substantially hinders dimerization. The use of a mutant glutathione S-transferase which retains activity yet has a greatly weakened tendency to dimerize (such as R69E) may be advantageous for certain applications of GST fusion proteins.