G protein subunit association and dissociation are thought to play an important role in signal transduction. We measured alpha beta gamma heterocomplex formation using resonance energy transfer. Fluorescein-labelled alpha(F-alpha) emission was quenched approximately 10% on mixing with eosin-labelled beta gamma(E-beta gamma). Unlabelled beta gamma did not quench F-alpha fluorescence. Stopped-flow kinetics showed a t1/2 ranging from 2.5 s to 0.25 s for 50 nM to 1200 nM E-beta gamma. The rate saturated at high E-beta gamma concentrations consistent with a two-step mechanism. We report the first rapid-mix studies of G protein subunit association kinetics which suggest that alpha and beta gamma combine by a two-step process with a maximal rate of 4.1 +/- 0.4 s-1.