Random chemical mutagenesis, in vitro, of the 5′ portion of the Escherichia coli trpA gene has yielded 66 mutant α subunits containing single amino acid substitutions at 49 different residue sites within the first 121 residues of the protein; this portion of the α subunit contains four of the eight α helices and three of the eight β strands in the protein. Sixty-two of the subunits were examined for their heat stabilities by sensitivity to enzymatic inactivation (52 °C for 20 min) in crude extracts and by differential scanning calorimetry (DSC) with 29 purified proteins. The enzymatic activities of mutant α subunits that contained amino acid substitutions within the α and β secondary structures were more heat labile than the wild-type α subunit. Alterations only in three regions, at or immediately C-terminal to the first three β strands, were stability neutral or stability enhancing with respect to enzymatic inactivation. Enzymatic thermal inactivation appears to be correlated with the relative accessibility of the substituted residues; stability-neutral mutations are found at accessible residual sites, stability-enhancing mutations at buried sites. DSC analyses showed a similar pattern of stabilization/destabilization as indicated by inactivation studies. Tm differences from the wild-type α subunit varied ± 7.6 °C. Eighteen mutant proteins containing alterations in helical and sheet structures had Tm's significantly lower (-1.6 to -7.5 °C) than the wild-type Tm (59.5 °C). In contrast, 6 mutant α subunits with alterations in the regions following β strands 1 and 3 had increased Tm's (+1.4 to +7.6 °C). Because of incomplete thermal reversibilities for many of the mutant α subunits, most likely due to identifiable aggregated forms in the unfolded state, reliable differences in thermodynamic stability parameters are not possible. The availability of this group of mutant α subunits which clearly contain structural alterations should prove useful in defining the roles of certain residues or sequences in the unfolding/folding pathway for this protein when examined by urea/guaninidine denaturation kinetic analysis. © 1992.