Activating TSH receptor (TSHR) mutations are a major cause of toxic thyroid adenoma and familial hyperthyroidism and more than 37 such mutations have been described. Previously their functional activity has been assessed in terms of cAMP and inositol phosphate production and predominantly in transiently transfected COS-7 (monkey embryonic kidney cells), a model which does not reflect effects on thyrocyte proliferation and function. Here we have performed a systematic comparison of wild-type and 7 gain-of-function TSHR mutants, introduced into rat FRTL-5 and human thyrocytes, using retroviral vectors. Our results show that 1. biological potency of TSHR mutants in thyroid cells does not correlate with their cAMP levels in transfected COS cells highlighting the importance of cellular context and level of expression when assessing biological effects of oncogenic mutations; 2. dissociation between stimulation of function and growth occurs with thyrocyte differentiated functions more readily stimulated than growth; 3. TSHR mutants show a similar 'order of potency' in FRTL-5 cells and human thyrocytes; 4. mutants inducing the highest stimulation of adenylyl cyclase may paradoxically fail to induce proliferation and 5. biological effects of cAMP activating TSHR mutants are attenuated by complex counter-regulatory mechanisms at least at the level of phosphodiesterases and CREM isoforms.