Despite the growing structural information on receptors and G proteins, the information on affinities and kinetics of protein-protein and protein-nucleotide interactions is still not complete. In this study on photoactivated rhodopsin (R*) and the rod G protein, G(t), we have used kinetic light scattering, backed by direct biochemical assays, to follow G protein activation. Our protocol includes the following: (i) to measure initial rates on the background of rapid depletion of the G(t)GDP substrate; (ii) to titrate G(t)GDP, GTP, and GDP; and (iii) to apply a double displacement reaction scheme to describe the results. All data are simultaneously fitted by one and the same set of parameters. We obtain values of K(m) = 2200 G(t)/microm(2) for G(t)GDP and K(m) = 230 microm for GTP; dissociation constants are K(d) = 530 G(t)/microm(2) for R*-G(t)GDP dissociation and K(d) = 270 microm for GDP release from R*G(t)GDP, once formed. Maximal catalytic rates per photoexcited rhodopsin are 600 G(t)/s at 22 degrees C and 1300 G(t)/s at 34 degrees C. The analysis provides a tool to allocate and quantify better the effects of chemical or mutational protein modifications to individual steps in signal transduction.