Our previous in vivo studies demonstrated that thyroid hormone promotes the expression of the fibronectin (FN) gene in the rat liver, while it inhibits the synthesis in cultured human skin fibroblasts. These results can be interpreted as either different regulation of FN synthesis or gene expression among tissues, or divergent results of experiments performed in vivo or in vitro. Here we report on the action of thyroid hormone on FN gene expression in vitro using primary cultured hepatocytes compared to that in cultured skin fibroblasts. Hepatocytes were isolated from hypothyroid rats and were cultured in medium supplemented with thyroidectomized bovine serum (TxBS) or fetal bovine serum (FBS). T3 was added 2 or 24 h after plating, and cells were harvested after 2, 6, or 24 h. Total RNA was extracted, and mRNAs for rat FN and albumin were measured. The requirement of de novo protein synthesis for thyroid hormone-mediated induction of FN mRNA was examined by the addition of cycloheximide 15 min before T3 addition. The amount of FN mRNA significantly decreased in the hepatocytes cultured with TxBS compared with those cultured with FBS. The addition of T3 to TxBS resulted in the restoration of FN mRNA to the level in hepatocytes cultured in FBS. FN mRNA increased during the course of culture in the absence of T3; however, a further increase was observed 6 h after T3 addition. The abundance of albumin RNA decreased during the course of culture, but unlike FN mRNA, it was not changed by T3 addition. The increase in FN mRNA by T3 was not influenced by cycloheximide. These results indicate that thyroid hormone enhances FN gene expression in hepatocytes by its direct action without requiring de novo protein synthesis. In contrast, T3 decreased FN mRNA in cultured skin fibroblasts. Thus, the mode of thyroid hormone action on FN gene expression is different among tissues.