Antigenic sites for six monoclonal antibodies that bind to the alpha subunit (G alpha) of the photoreceptor guanyl nucleotide-binding protein (G-protein or transducin) have been determined. Five of these antibodies (4A, 7A, 7B, 7C, and 7D) were shown in the preceding paper (Hamm, H. E., Deretic, D., Hofmann, K. P., Schleicher, A., and Kohl, B. (1987) J. Biol. Chem. 262, 10831-10838) to block G-protein-rhodopsin interaction. We have blotted tryptic and chymotryptic peptides of G-protein to nitrocellulose paper and found that these antibodies bind to peptides that contain the COOH-terminal end of the protein assessed by 32P-ADP-ribosylation of the COOH-terminus by pertussis toxin. The antigenic site is not exactly at the COOH-terminus since the antibodies also bind two peptides which lack a 2-kDa piece from the COOH-terminus. Antigenic sites are therefore on the 7-kDa chymotryptic peptide and 5-kDa tryptic peptide more than 2 kDa away from the COOH-terminus. Further evidence for this antigenic site comes from the ability of these antibodies to block pertussis toxin-mediated ADP-ribosylation while still binding to the previously ADP-ribosylated protein both on nitrocellulose blots and in immunoprecipitations. Antibody 4H, which was shown not to interrupt any of the functions studied, binds to the 11-kDa major tryptic fragment. To aid in the mapping of these sites onto the surface of G alpha, a model of the three-dimensional structure of G alpha has been generated using the G alpha primary sequence, predicted secondary structure, hydropathy plot, and the constraints of the GDP-binding site of the GTP-binding protein elongation factor Tu solved by Jurnak (Jurnak, F. (1985) Science 230, 32-36).