In the dark, the activity of the rod cGMP phosphodiesterase (PDE) catalytic alpha- and beta-subunits (P alpha beta) is inhibited by two gamma-subunits (P gamma). On light stimulation of the photoreceptor cells, the GTP-bound alpha-subunit of visual G-protein transducin (GtaGTP) displaces the P gamma-subunits from their inhibitory sites on P alpha beta, leading to the effect or enzyme activation. We designed a number of P gamma mutants, each with a single cysteine residue evenly distributed at a different position along the P gamma polypeptide chain. These cysteine residues served as sites for the introduction of the environmentally sensitive fluorescent probe, 3-(bromoacetyl)-7-diethyl aminocoumarin (BC). Analysis of the interactions of P alpha beta and Gta with the fluorescently labeled P gamma mutants suggests two distinct functional interfaces of P gamma. The P alpha beta/P gamma interface is formed essentially by the C-terminus of P gamma and by the N-terminal portion of the P gamma polycationic region, P gamma-24-45, whereas the P gamma/Gta interface includes the C-terminal portion of P gamma-24-45 and the region surrounding P gamma Cys68. Such functional organization of P gamma may represent an important element for the PDE activation mechanism during transduction of visual signals.