The functional significance of residues in the RecA protein P-loop motif was assessed by analyzing 100 unique mutants with single amino acid substitutions in this region. Comparison of the effects on the LexA coprotease and recombination activities shows that Pro67 is unique among these residues because only at this position did we find substitutions that caused differential effects on these functions. One mutant, Pro67-->Trp, displays high constitutive coprotease activity and a moderate inhibitory effect on recombination functions. Glu and Asp substitutions result in low level constitutive coprotease activity but dramatically reduce recombination activity. The purified Pro67-->Trp protein shows a completely relaxed specificity for NTP cofactors in LexA cleavage assays and can use shorter length oligonucleotides as cofactors for cleavage of lambda cI repressor than can wild type RecA. Interestingly, both the mutant protein and wild type RecA can use very short oligonucleotides, e.g. (dA)6 and (dT)6, as cofactors for LexA cleavage. We have also found two mutations at position 67, which are completely defective for LexA coprotease activity in vivo but still maintain recombinational DNA repair (Pro67-->Lys) and homologous recombination (Pro67-->Lys and Pro67-->Arg) activities. These findings show that the recombination activities of RecA are mutationally separable from the coprotease function and that Pro67 is located in a functionally important position in the RecA structure.