We have used site-directed mutagenesis to replace amino acid 1 of the recognition alpha-helix of the catabolite gene activator protein (CAP), Arg-180, with glycine and with alanine. Substitution of Arg-180 of CAP eliminated specificity between G.C, A.T, C.G, and T.A at base pair 5 of the DNA half-site. The effect was position-specific: substitution of Arg-180 did not eliminate specificity between G.C, A.T, C.G, and T.A at base pair 7 of the DNA half-site. We conclude, in agreement with the model for the structure of the CAP-DNA complex [Weber, I. & Steitz, T. (1984) Proc. Natl. Acad. Sci. USA 81, 3973-3977; and Ebright, R., Cossart, P., Gicquel-Sanzey, B. & Beckwith, J. (1984) Proc. Natl. Acad. Sci. USA 81, 7274-7278], that Arg-180 of CAP makes a specificity-determining contact with base pair 5 of the DNA half-site in the CAP-DNA complex. The identification of the contact by Arg-180 in this report, in conjunction with the identification of the contact by Glu-181 in a previous report [Ebright, R., Cossart, P., Gicquel-Sanzey, B. & Beckwith, J. (1984) Nature (London) 311, 232-235], provides information sufficient to define the orientation of the helix-turn-helix motif of CAP with respect to DNA in the CAP-DNA complex.