Many GPCRs are now recognized to trigger a second phase of G protein signaling from endosomes. The presence of activated GPCRs on endosomes has been clearly shown, especially among Gs-coupled GPCRs, but endosomal signaling ultimately requires activation of the G protein, which mediates downstream signal transduction. How GPCRs regulate both G protein abundance and G protein activation on endosomes is incompletely understood. In this dissertation, I address this question by dissecting the regulation of GPCR-triggered G protein localization to and activation on endosomes. I first verify that activation of a model GPCR, the β2AR, at the plasma membrane drives translocation of Gαs to endosomes separately from trafficking of the activated receptor. Focusing on VIPR1, a GPCR that produces a well-resolved endosomal signal, I demonstrate the presence of active-state, endogenous Gαs at both the plasma membrane and endosomes, and I show that Gαs activation on endosomes is dependent on receptor endocytosis. I also detect VIPR1-mediated stimulation of an additional component of endogenous G protein activity, which I ascribe to Gαq/11, consistent with the known dualcoupling ability of VIPR1. Interestingly, VIPR1 preferentially activates Gαq/11 on the plasma membrane but Gαs on endosomes, and I further show location-specific preferences in Gαs and Gαq/11 activation by a second GPCR, the A2BR. Next, I investigate the mechanisms of reversible Gαs redistribution to endosomes. I confirm that Gαs dissociates from the plasma membrane after GPCR activation, samples endosomes and additional internal compartments, and returns to the plasma membrane after GPCR inactivation through a non-vesicular pathway. My results provide xii critical insights into the subcellular organization of G protein activation. Additionally, they uncover a new layer of location bias in cellular GPCR signaling, defined by changes in GPCR-G protein coupling at different subcellular locations.