In photoreceptor cells of vertebrates, the GTP-bound a-subunit of rod G-protein, transducin (Gta), interacts with the cGMP phosphodiesterase inhibitory c-subunit (Pc) to activate the effector enzyme. The GDP-bound Gta can also bind the Pc subunit, albeit with a lower affinity than GtaGTP. In this work, interactions between GtaGDP and Pc or Pc-24-45Cys labeled with the fluorescent probe 3-(bromoacetyl)-7-(diethylamino)coumarin (PcBC, Pc-24-45BC) have been investigated. Addition of GtaGDP to PcBC produced approximately a 6-fold maximal increase in the probe fluorescence, while the fluorescence of Pc-24-45BC was enhanced by 2.3-fold. The Kd's for the GtaGDP binding to PcBC and Pc-24-45BC were 75 ± 8 nM and 400 ± 110 nM, respectively. The Gtbc subunits had no notable effect on the binding of GtaGDP to PcBC or Pc-24-45BC, suggesting that Pc and Gtbc bind to GtaGDP noncompetitively. The Gtabc interaction with the fluorescently labeled Pc was effectively blocked in the light-activated rhodopsin (R*)-Gtabc complex. Furthermore, addition of excess Pc or Pc-24-45 prevented binding of Gtabc to R*, indicating that the R* and Pc binding surfaces on Gtabc may overlap. It is likely that R* has a binding site within the a3-b5 region of Gta, which is a proposed site of GtaGDP binding to Pc-24-45. Alternatively, R* may induce conformational changes of the Gta a3-b5 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. Copyright 1997, American Chemical Society.