Three BODIPY GTPgammaS analogs (FL, 515, and TR), BODIPY FL GppNHp and BODIPY FL GTP molecules were synthesized as possible fluorescent probes to study guanine nucleotide binding spectroscopically. Binding to G(alphao) increases baseline analog fluorescence by 6-, 8.5-, 2.8-, 3.5-, and 3.0-fold, respectively. Binding of GTPgammaS and GppNHp analogs to G(alphao) is of high affinity (K(D) 11, 17, 55, and 110 nM, respectively) and reaches a stable plateau while fluorescence of BODIPY FL GTP shows a transient increase which returns to baseline. Furthermore, BODIPY FL GTPgammaS shows varying affinities for alpha(o), alpha(s), alpha(i1), and alpha(i2) (6, 58, 150, and 300 nM). The affinities of BODIPY FL GppNHp for all four G(alpha) subunits are 10-fold lower than for BODIPY FL GTPgammaS. Half-times for the fluorescence increase are consistent with known GDP release rates for those proteins. Enhancement of fluorescence upon binding the G(alpha) subunit is most likely due to a rotation around the gamma-thiol (GTPgammaS) or the 3' ribose-hydroxyl (GppNHp) bond to relieve the quenching of BODIPY fluorescence by the guanine base. Binding to G(alpha) exposes the BODIPY moiety to the external environment, as seen by an increase in sodium iodide quenching. The visible excitation and emission spectra and high fluorescence levels of these probes permit robust real-time detection of nucleotide binding.