We have examined the effect of triiodothyronine (T3) on de novo transformation of a cloned population of Fischer rat embryo fibroblasts (CREF) by a temperature-sensitive mutant (H5ts125) of type 5 adenovirus and on the expression of the transformed phenotype in these cells. When CREF cells were grown in medium lacking T3 before, during, and after infection with H5ts125, the yield of transformed foci was half that in the cultures supplemented with 1 nM T3. Selective addition or removal of T3 during various phases of the transformation process indicated that the hormone exerted its maximal effect within 72 hr after viral infection. T3 was also required for optimal growth in agar of two clones of CREF cells previously transformed by type 5 adenovirus, wt-3A and ts-7E. The tumor promoter 12-O-tetradecanoylphorbol 13-acetate could substitute for T3 in enhancing growth in agar of wt-3A but not of ts-7E, suggesting that the promoter and T3 modify anchorage-independent growth by different mechanisms. Normal CREF cells and both of the transformed CREF clones grew equally well in monolayer culture in medium containing or lacking T3. Both of the transformed CREF clones contained a lower number of nuclear T3 receptors than did CREF cells and they bound somewhat lower levels of phorbol dibutyrate. These results indicate that thyroid hormone modulates an early stage involved in adenovirus transformation and that it also enhances the expression of the transformed state in previously transformed cells.