The stimulatory G protein (Gs) mediates activation of adenylylcyclase by a ligand-receptor complex. Gs is heterotrimeric (alpha beta gamma) and activation can be accomplished by dissociation of the alpha-subunit (Gs alpha) from the beta gamma-subunit complex (G beta gamma). Gs alpha is also a substrate for choleragen catalyzed ADP-ribosylation when it is associated with G beta gamma but not as free Gs alpha. Using recombinant DNA techniques we modified the cDNA for the 52,000 M(r) form of Gs alpha (Gs alpha 52) to produce a protein with a 2,400 M(r) N-terminal extension (Gs alpha 54.4). This N-terminal extension could be removed with the protease Factor Xa. In vitro transcription and translation of the recombinant plasmid containing the cDNA's for Gs alpha 52 and Gs alpha 54.4 produced a 52,000 M(r) and a 54,000 M(r) protein, respectively. In solution the properties of Gs alpha 52 and Gs alpha 54.4 were indistinguishable. Both proteins: (a) formed a heterotrimer with G beta gamma and their affinities for the subunit complex were the same; (b) could be ADP-ribosylated by choleragen in the presence but not in the absence of G beta gamma; (c) bound the non-hydrolyzable GTP analogue, GTP gamma S, and were protected from chymotryptic proteolysis by the guanine nucleotide; and (d) could activate in vitro translated type IV adenylylcyclase. Gs alpha 54.4 and Gs alpha 52 were incorporated into S49 cyc-membranes, which lack Gs alpha. After incorporation, both Gs alpha 52 and Gs alpha 54.4 were protected from chymotryptic proteolysis when GTP gamma S was present, revealing that both proteins were able to bind the nucleotide and undergo a conformational change characteristic of Gs alpha activation. When Gs alpha 52 was incorporated into cyc-membranes it could mediate both hormone and GTP gamma S stimulation of adenylylcyclase and could be ADP-ribosylated by choleragen, but Gs alpha 54.4 could do neither of these things, indicating that the properties of Gs alpha 54.4 were altered by the membrane. Deletion of the N-terminal extension by treatment with Factor Xa in solution converted Gs alpha 54.4 to Gs alpha 52, and upon incorporation into cyc-membranes it behaved like Gs alpha 52 in every regard, showing that the effect of the N-terminal extension was reversible. A lack of other differences in the functional properties of Gs alpha 52 and Gs alpha 54.4 suggests a correlation between the interaction of Gs alpha with G beta gamma and its ability to activate adenylylcyclase.