The interaction of six hydrolysis-resistant analogues of GTP with transducin, the signal-coupling protein in vertebrate photoreceptors, was investigated. GppNHp and GppCH2p differ from GTP at the bridging position between the beta- and gamma-phosphate groups. The other analogues studied (GTP gamma F, GTP gamma OMe, GTP gamma OPh, and GTP gamma S) differ from GTP in containing a substituent on the gamma-phosphorus atom or at a nonbridging gamma-oxygen atom. Competition binding experiments were carried out by adding an analogue, [alpha-32P]GTP, and a catalytic amount of photoexcited rhodopsin (R) to transducin and measuring the amount of bound [gamma-32P]GTP. The order of effectiveness of these analogues in binding to transducin was GTP gamma S greater than GTP much greater than GppNHp greater than GTP gamma OPh greater than GTP gamma OMe greater than GppCH2p greater than GTP gamma F A second assay measured the effectiveness of GTP gamma S, GppNHp, and GppCH2p in eluting transducin from disc membranes containing R. The basis of this assay is that transducin is released from disc membranes when it is activated to the GTP form. The relative potency of these three analogues in converting transducin from a membrane-bound to a soluble form was 1000, 75, and 1, respectively. Stimulation of cGMP phosphodiesterase activity served as a third criterion of the interaction of these analogues with transducin. The order of effectiveness of these analogues in promoting the transducin-mediated activation of the phosphodiesterase was GTP gamma S greater than GTP much greater than GppNHp greater than GTP gamma OPh much greater than GppCH2p greater than GTP gamma OMe greater than GTP gamma F GTP gamma S was more than a 1000 times as potent as GTP gamma F in activating the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)