Rat thyroid cells (FRTL5), transfected with the sequence coding for rat insulin-like growth factor II (IGF-II) presented mRNA specific for the transfected IGF-II in most of the clones obtained (Tr clones). Tr7 and Tr12 cells maintained their ability to respond to the mitogenic effect of thyrotropin (TSH), while either exogenous IGF-I or IGF-II or insulin failed to stimulate their proliferation. In the absence of exogenous mitogens the Tr7 and Tr12 clones vigorously incorporated [3H]thymidine into DNA. This activity was significantly inhibited by sm1.2, a monoclonal antibody against rat IGF-II. Tr7 and Tr12 clones possess type I IGF receptors, known to mediate the mitogenic effect of IGF-II, with affinity similar to those present on the membrane of the parental cells but with reduced capacity. Finally, media conditioned by Tr7 and Tr12 increase basal thymidine incorporation in quiescent FRTL5 cells and amplify that induced by TSH. Endogenous IGFs may play an important role in the regulation of thyroid cell proliferation by modulating the mitogenic effect of TSH and by supporting TSH-independent growth.