When primary cultures of rat hepatocytes were placed in a chemically defined serum-free medium containing a combination of insulin, glucagon, and dexamethasone, the synthesis of albumin and total protein and the cellular content of RNA and DNA were maintained at constant values for 8 days. Despite the constant rate of albumin synthesis, secretion of the protein increased more than twofold during the initial 4 days in culture and was then maintained at a value similar to that observed in vivo through day 8. This observation suggested an initial defect in albumin secretion that was corrected with time in culture. Deprivation of insulin between days 2 and 5 resulted in a decline in albumin secretion to approximately 40% of the control value. The decline in albumin secretion was accompanied by proportional decreases in albumin synthesis, albumin mRNA, and albumin gene transcription. Return of insulin-deprived cells to complete medium on day 5 restored albumin synthesis and secretion as well as albumin mRNA to control values by day 8. Deprivation of either glucagon or dexamethasone also resulted in reduced albumin synthesis and secretion accompanied by proportional decreases in albumin mRNA and gene transcription. However, the magnitude of the changes in these parameters was less with glucagon or dexamethasone deprivation compared with insulin deprivation. Return of glucagon- or dexamethasone-deprived cells to complete medium on day 5 restored albumin synthesis and secretion as well as albumin mRNA to control values by day 8.(ABSTRACT TRUNCATED AT 250 WORDS)