To further our studies on collagen gene expression, we have evaluated the molecular basis for the finding that steroids decrease collagen synthesis in cultured hepatocytes. We studied the effects of dexamethasone on primary cultures of adult rat hepatocytes grown on tissue culture plastic in either serum-supplemented medium or a serum-free hormonally defined medium. Cells were plated and allowed to attach for 24 hr in a mixture of serum-supplemented medium + hormonally defined medium. Cultures were then fed every 24 hr for 4 days under 1 of 4 conditions: serum-supplemented medium, serum-supplemented medium + dexamethasone, hormonally defined medium or hormonally defined medium + dexamethasone. On the fifth day, RNA was extracted. Dexamethasone did not affect the amount of RNA isolated; nor did it influence the quantitative translation of the mRNA in the rabbit reticulocyte lysate mRNA-dependent system. Employing hybridization analysis, dexamethasone resulted in increased albumin mRNA content in hepatocytes grown in serum-supplemented medium but had no affect on hormonally defined medium, and decreased type I in collagen mRNA content in cells grown in either serum-supplemented medium or hormonally defined medium. In cells cultured in hormonally defined medium, the β-actin and procollagen mRNA levels were lower than those in serum-supplemented medium, but albumin mRNA levels were higher, and in fact equivalent to those in vivo. β-Actin mRNA levels were not affected by dexamethasone in either serum-supplemented medium or hormonally defined medium. These results suggest that hormonally defined medium improves the expression of tissue-specific functions in hepatocytes, and dexamethasone reduces Type I collagen mRNA content in hepatocytes as well as mesenchymal cells. It is presently not clear whether the changes in albumin and collagen steady state mRNA levels are due to transcriptional and/or post-transcriptional controls.