The mechanism of interaction of the G-protein of retinal rods with rhodopsin and with nucleotides has been investigated using two independent techniques, light-scattering and direct binding measurements with labeled nucleotides. Binding of photoexcited rhodopsin (R*) and nucleotides are shown to be antagonist, and three conformations of the G-protein are described, each of which is proposed to be related to a different level of light-scattering, as follows: (a) the "dark" state, stable in the absence of photoexcited rhodopsin, in which the nucleotide site is poorly accessible and has a high affinity (dissociation constants, 0.1 microM for GDP and 0.01 microM for GppNHp); (b) the R*-bound state in which the nucleotide site is rapidly accessible with a lower affinity (dissociation constants, about 20 microM for GDP and GTP; 20-100 microM for GppNHp). Binding of R* to the G-protein therefore enables rapid binding or exchange of the nucleotide; this in turn reduces the affinity of the G-protein for R* (dissociation constants, 0.2 microM for G-protein with GDP bound and 2-10 microM for G-protein with GppNHp bound, compared to 1 nM in absence of bound nucleotide); and (c) the third state, the activator of the phosphodiesterase. In the presence of GTP, an additional irreversible and fast step, which is proposed to be the dissociation of alpha-GTP from beta gamma, is shown to occur; a steady state equilibrium is obtained, and the dissociation constant measured between GTP and this third state of the G-protein in the presence of R* is an apparent constant which depends on the rate of transconformation between the first two states and on the rate of GTP hydrolysis. The minimum value of this apparent dissociation constant for GTP (0.05-0.1 (microM) is obtained at high levels of illumination. Finally, some results (number of nucleotide sites and saturation of the rate of the light-scattering signal) suggest an oligomeric association of the G-protein.