We have characterized about 50 different amino acid substitutions in the aminoterminal domain of lambda repressor. Sixteen of these substitutions alter external side chains of the repressor and cause a substantial reduction in the affinity of the mutant repressor for operator DNA. Seven of these mutant repressors were purified and were shown to be stably folded. The strong, external repressor mutations occur near the aminoterminal end of alpha helix 2, throughout alpha helix 3, and in the aminoterminal-arm region of the repressor. These results suggest that these regions of lambda repressor are close to operator DNA in the protein-DNA complex and thus that these regions comprise the DNA-binding sites of the repressor. Our genetic results support and are completely consistent with more-detailed models of the repressor-operator interaction based on model-building (Pabo and Lewis 1982; Lewis et al. this volume) and biochemical studies (Pabo et al. 1982).