In photoreceptor cells of vertebrates, the GTP-bound alpha-subunit of rod G-protein, transducin (G(t alpha)), interacts with the cGMP phosphodiesterase inhibitory gamma-subunit (Pgamma) to activate the effector enzyme. The GDP-bound G(t alpha) can also bind the Pgamma subunit, albeit with a lower affinity than G(t alpha)GTP. In this work, interactions between G(t alpha)GDP and Pgamma or Pgamma-24-45Cys labeled with the fluorescent probe 3-(bromoacetyl)-7-(diethylamino)coumarin (PgammaBC, Pgamma-24-45BC) have been investigated. Addition of G(t alpha)GDP to PgammaBC produced approximately a 6-fold maximal increase in the probe fluorescence, while the fluorescence of Pgamma-24-45BC was enhanced by 2.3-fold. The Kd's for the G(t alpha)GDP binding to PgammaBC and Pgamma-24-45BC were 75 +/- 8 nM and 400 +/- 110 nM, respectively. The G(t betagamma) subunits had no notable effect on the binding of G(t alpha)GDP to PgammaBC or Pgamma-24-45BC, suggesting that Pgamma and G(t betagamma) bind to G(t alpha)GDP noncompetitively. The G(t alpha betagamma) interaction with the fluorescently labeled Pgamma was effectively blocked in the light-activated rhodopsin (R*)-G(t alpha betagamma) complex. Furthermore, addition of excess Pgamma or Pgamma-24-45 prevented binding of G(t alpha betagamma) to R*, indicating that the R* and Pgamma binding surfaces on G(t alpha betagamma) may overlap. It is likely that R* has a binding site within the alpha3-beta5 region of G(t alpha), which is a proposed site of G(t alpha)GDP binding to Pgamma-24-45. Alternatively, R* may induce conformational changes of the G(t alpha) alpha3-beta5 region such that the resulting structural changes alter the adjacent consensus sequence for the guanine ring binding of GDP/GTP(NKXD), and lead to a reduction in the affinity of G-protein for guanine nucleotides.