Although many G protein-coupled receptors (GPCRs) are known to activate multiple signaling pathways by coupling to different types of G proteins or by promoting G protein-independent events, how this occurs remains unclear. Using bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer, we provide evidence for protease-activated receptor 1 (PAR(1)) forming preassembled complexes with Galphai1 but not Galpha12. PAR(1) activation appears to rapidly induce transient Galphai1 activation (t(1/2) = 4.13 s) but late and stable recruitment of Galpha12 (t(1/2) = 8.8 min) in parallel with beta-arrestin 1 (t(1/2) = 7.5 min). However, there is no significant difference in the potency of the agonist-dependent response between Galphai1, Galpha12, and beta-arrestin 1 (EC(50) values 0.48, 0.30, and 0.15 nM, respectively). Although it seems beta-arrestin 1 is recruited to preassembled PAR(1)-Galphai1 complexes, this appears unlikely with Galpha12, suggesting 2 distinct receptor populations. Of note, we observed a different Galpha12 association mode with other GPCRs, indicating that preassembly and association dynamics may be specific properties of a receptor-G protein pair. Furthermore, the Galpha C terminus appears to play different roles in the distinct association modes. Consequently, G protein preassembly or recruitment may constitute novel mechanisms for controlling the kinetics and multitude of GPCR signaling pathways.