The kinetic constants for the ADP-ribosylation of transducin were determined for the recombinant S1 subunit of pertussis toxin (rS1, composed of 235 amino acids) and two genetically derived deletion peptides, C180 and C195, which are composed of the 180 and 195 amino-terminal residues of the S1 subunit, respectively. Titration of NAD in the presence of a constant concentration of transducin (0.5 microM) showed that the KmappNAD in the ADP-ribosylation of transducin were similar, approximately 20 microM, for rS1, C195, and C180. In contrast, titration of transducin in the presence of a constant concentration of NAD (25 nM) showed that rS1 possessed a lower Kmapp(transducin) and greater kcat than either C195 or C180. Previous studies (Cortina, G., and Barbieri, J.T. (1991) J. Biol. Chem. 266, 3022-3030) showed that the 16 carboxyl terminal residues of the S1 subunit did not function in the ADP-ribosylation of transducin. It thus appears that residues between 195 and 219 of the S1 subunit are required for high affinity transducin binding and may be involved in the transfer of ADP-ribose to transducin. To localize the defect in the recognition of transducin by C180, rS1 and C180 were assayed for the ability to ADP-ribosylate either transducin or the purified alpha subunit of transducin (T alpha). Upon saturation of the target protein, rS1 ADP-ribosylated equivalent moles of transducin or T alpha, with the linear velocity of rS1-mediated ADP-ribosylation of transducin approximately 16-fold more rapid than the rate of ADP-ribosylation of T alpha. In contrast, the initial linear velocity of C180-mediated ADP-ribosylation of transducin was only 1.7-fold more rapid than the rate of ADP-ribosylation of T alpha. These data indicate that the amino-terminal 180 amino acids of S1 confer the specificity for ADP-ribosylation primarily through the interaction with T alpha, while residues between 195 and 219 of S1 confer high affinity binding to transducin primarily through the interaction, either directly or indirectly, with T beta gamma.