ABSTRACT The thyroid‐stimulating hormone (TSH) receptor signals via G s to produce cAMP and via G q/11 to produce inositol‐1,4,5‐trisphosphate, which is degraded to inositol monophosphate (IP1; phosphoinositide signaling). The potency of TSH for cAMP signaling is higher than for phosphoinositide signaling, and it was suggested that there are “spare receptors” for cAMP signaling. In a human embryonic kidney macrophage scavenger receptor‐expressing (HEK‐EM) 293 model system, there are no spare receptors, but the cells still exhibited 100‐fold differences in potencies. Dose responses for TSH‐stimulated dissociation of prebound 125 I‐TSH (negative cooperativity; EC 50 =70 mU/ml), which requires TSH binding to both sites of the TSH receptor (TSHR) homodimer, and TSH‐stimulated IP1 production (EC 50 =50 mU/ml) were indistinguishable. Fluorescence resonance energy transfer (FRET) using tagged receptors showed that TSHR formed homodimers and heterodimers with two binding‐deficient mutant TSHRs, L252P and C41S. When L252P or C41S was expressed with TSHR, that is, when TSHR/L252P or TSHR/C41S heterodimers could only bind one TSH, TSH‐stimulated IP1 production was decreased relative to cAMP production. The slopes of linear regression analyses comparing fold stimulation by TSH of IP1 vs. cAMP production were 0.044 ± 0.0047, 0.0043 ± 0.0041, and 0.0059 ± 0.0014 for cells expressing TSHR alone, TSHR and L252P, or TSHR and C41S, respectively. We suggest that TSHR coupling to phosphoinositide signaling is dependent on binding 2 molecules of TSH to TSHR homodimer, causing a conformational change allowing coupling to G q/11 .—Allen, M. D., Neumann, S., Gershengorn, M. C. Occupancy of both sites on the thyrotropin (TSH) receptor dimer is necessary for phosphoinositide signaling. FASEB J. 25, 3687–3694 (2011). www.fasebj.org