cDNAs for thyroid peroxidase (TPO) and thyroglobulin (Tg) have been cloned and sequenced. Using such cDNAs, we investigated the regulation of TPO and Tg gene expression by various agents in cultured human thyroid cells. Unstimulated human thyroid cells contained a major RNA species [3.2 kilobases (kb) in length] and several minor RNA species (less than 3.2 kb mRNA) of TPO, but no detectable Tg transcripts. However, TSH-stimulated thyroid cells contained four distinct TPO mRNA species (4.0, 3.2, 2.1, and 1.7 kb) and a single Tg mRNA species (8.5 kb). TSH stimulated the TPO and Tg mRNA levels in a dose- and time-dependent manner. The same results were obtained with 8-bromo-cAMP, a cAMP analog, but not with insulin or insulin-like growth factor I. Furthermore, inductions of these mRNAs by TSH and 8-bromo-cAMP were almost completely blocked by cycloheximide, a protein synthesis inhibitor. These results suggest that human thyroid cells contain four distinct TPO mRNAs and a single species of Tg mRNA, and the levels of all mRNAs are increased by TSH/cAMP stimulation. These increases are blocked by inhibiting protein synthesis, indicating that TSH stimulation of TPO and Tg mRNA levels may be mediated by newly synthesized protein(s).