In vitro mutagenesis and transient expression in COS cells has been used to associate a missense mutation with a clinical or biochemical phenotype. Mutations affecting the alpha-subunit of beta-hexosaminidase A (alpha beta) (E.C.3.2.1.52) result in Tay-Sachs disease. Because hexosaminidase A is heterodimeric, analysis of alpha-chain mutations is not straightforward. We examine three approaches utilizing previously identified mutations affecting alpha-chain folding. These involve transfection of (1) the alpha cDNA alone; (2) a beta cDNA construct encoding a beta-subunit substituted at a position homologous to that of the alpha-subunit, and (3) both alpha and beta cDNAs. The latter two procedures amplified residual activity levels over that of patient samples, an effect not previously found with mutations affecting an "active" alpha Arg residue. This effect may help to discriminate between protein-folding and active-site mutations. We conclude that, with proper controls, the latter method of cotransfection can be used to evaluate the effects and perhaps to predict the clinical course of some alpha-chain mutations. Using this technique, we demonstrate that the adult-onset Tay-Sachs mutation, alpha Gly --> Ser269, does not directly affect alpha beta dimerization but exerts an indirect effect on the dimer through destabilizing the folded alpha-subunit at physiological temperatures. Two other alpha mutations linked to more severe phenotypes appear to inhibit the initial folding of the subunit.