The limited proteolytic pattern of transducin, Gt, and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the alpha t subunit were identified. The alpha t subunit in the GTP gamma S bound form was cleaved into a major 38 kD fragment, whereas alpha t-GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The beta gamma t subunit was not very sensitive to proteolytic digestion with chymotrypsin. The gamma t subunit was not cleaved and only a small portion of beta t was digested into several fragments. In order to determine which proteolytic fragment of alpha t still contained the carboxyl terminal region, chymotrypsinization was carried out using Gt previously 32P-labeled at Cys347 by pertussis toxin-catalyzed ADP-ribosylation. The 32P-label was mainly associated with the alpha t subunit and a 15 kD fragment. The 23 and 21 kD fragments were not 32P-labeled. Analysis of amino terminal sequences of 38, 21, and 15 kD proteolytic bands allowed the identification of the major cleavage sites. Chymotrypsin had two cleavage sites in the amino terminal region of alpha t, at Leu15 and Leu19. Chymotrypsin removed 15-19 amino acid residues from the amino terminus of alpha t, generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp207 in a conformation-dependent manner. Trp207 of alpha t-GTP gamma S was resistant to proteolysis but alpha t-GDP and the 38 kD fragments of alpha t-GDP produced the 23 and 21 kD fragments, respectively, and a 15 kD fragment containing the carboxyl terminus.(ABSTRACT TRUNCATED AT 250 WORDS)