The present study provides evidence that G protein coupled receptor (GPCR) signaling pathways participate in an interactive signaling network governed by the principles of mass action. Using an inducible thromboxane A2 receptor (TPR)/platelet activating factor receptor (PAFR) co-expressing cell model, TPR or PAFR expression was independently up-regulated. Immunostaining and radioligand binding experiments demonstrated that this receptor up-regulation resulted in increased GPCR:G protein mass ratios. This increase in mass ratio impacted both TPR and PAFR ligand affinity. Specifically, up-regulating TPR expression not only decreased TPR ligand affinity, but also decreased the ligand affinity of PAFRs. A similar effect on ligand affinities was observed when PAFRs were up-regulated. In addition, increasing the GPCR:G protein mass ratio for TPRs led to desensitization of the calcium mobilization response to PAFR activation, and increasing PAFR mass desensitized the TPR-mediated calcium response. Finally, it was observed that an increased TPR:G protein mass ratio was associated with a shift in the TPR signaling response, and revealed an additional TPR signaling pathway through G(S). Collectively, these results describe a novel mechanism, i.e., mass-dependent GPCR signaling, by which cells can modulate their GPCR signaling pathways and signaling priorities.