The regulatory effects of thyroid hormone on biosynthesis of myocardial proteins that originate from cardiac myocytes are well established. Little is known, however, of regulatory effects of thyroid hormone on interstitial proteins. In this study we examined the effects of thyroid hormone on collagen gene expression in thyroid hormone-induced myocardial hypertrophy and the response of cardiac fibroblasts to thyroid hormone in culture. Adult male Sprague-Dawley rats were treated intraperitoneally with L-thyroxin (10 μg/100 g body wt) for 2 hours or 1, 2, 3, 6, 12, or 14 days. Northern blot analysis of RNA from total ventricular tissue showed that after 2 hours of treatment, the abundance of mRNA for pro α2(I) collagen decreased by 53% (p<0.05) and reached the lowest level (60% decrease, p<0.02) at day 1, remained diminished at day 3, and then gradually returned toward normal levels. After transient transfection of chimeric DNA containing collagen type I promoter-chloramphenicol acetyl transferase (CAT) gene into the thyroxin-treated cardiac fibroblasts, the level of CAT activity decreased significantly. Treatment of cardiac fibroblasts in culture (10 nM l-thyroxin) resulted in a 33% (p< 0.005) decrease in the abundance of mRNA for pro α2(I) collagen. The stability of the mRNA for pro α2(I) collagen in cardiac fibroblasts, as measured by mRNA half-life, was slightly (16.6%) decreased by thyroid-hormone treatment. Collagen synthesis as shown by immunofluorescent staining of intracellular collagen in cultured fibroblasts and in frozen sections of myocardium was also diminished. Interestingly, the abundance of mRNA for transforming growth factor-β1in the myocardium increased by 53% (p<0.05) within hours and reached a peak (75% increase,p< 0.001) at day 1 after treatment. The abundance of mRNA for transforming growth factor-β1was also increased (94%,p< 0.005) in cardiac fibroblasts after treatment with l-thyroxin. Thyroxin treatment of cardiac fibroblasts led to the induction of proto-oncogenes c-fosand c-junand early growth response gene within 1 hour. Treatment of cardiac fibroblasts with l-thyroxin led to a 357% (p< 0.001) increase in [3H]thymidine incorporation into the cell nuclei compared with that in untreated cells. Together, these findings suggest that cardiac fibroblasts and proteins originating from them are targets for thyroid hormone action in the myocardium and that this hormone plays a regulatory role on interstitial protein gene expression. Data further suggest that thyroid hormone-induced myocardial hypertrophy could be distinguished by the lack of cardiac fibrosis.