Using a combinatorial cassette mutagenesis procedure we have introduced a large number of single and multiple amino acid substitutions into an area of the RecA protein defined by residues 152-159. This sequence overlaps the disordered loop 1 region (L1) in the RecA crystal structure which has been hypothesized to be involved in DNA binding. Assays for recombinational DNA repair and LexA coprotease activities identify Glu154 as the only one of these 8 residues which is critical to RecA function. Several other mutations observed at nearby residues support the identity of Glu154 as the most important of the 14 residues in the area defined by Pro151 to Met164. In addition, Gly157 and Glu158 appear to be hot spots for the occurrence of mutation-induced constitutive coprotease activity.