In human normal thyrocytes, the cAMP-responsive signaling pathway plays a central role in gene regulation, cell proliferation, and differentiation. Constitutive activation of the cAMP signal transduction system has been documented in thyroid autonomously hyperfunctioning adenomas in which activating mutations in either the TSH receptor gene or the Gsalpha protein gene (gsp oncogene) have been described. The molecular mechanism whereby cAMP induces thyrocyte proliferation is unknown, but recent evidence suggests that the transcription factor cAMP response element binding protein (CREB) may serve as an important biochemical intermediate in this proliferative response. Herein we have investigated the expression of CREB in normal and tumoral thyroid tissues from a series of ten unrelated patients with autonomously hyperfunctioning adenomas, previously screened for mutations in the TSH receptor and Gsalpha genes. In all tumors examined, the expression of the activated, phosphorylated form of CREB was markedly reduced compared with that of the corresponding paired normal thyroid tissue, and this reduction was independent of the presence of mutations in the TSH receptor gene and Gsalpha gene. Moreover, no correlation was observed in these tissues between CREB phosphorylation and either protein kinase A activity or protein phosphatase expression. Thus, these data suggest that in human hyperfunctioning thyroid adenomas, the PKA/CREB system does not play a role in cell proliferation.