Primary monolayer cell cultures of adult rat hepatocytes underwent change in morphology and substantial cell loss between 1 and 3 days postinoculation. Dexamethasone-supplementation (1 micronM) of the culture medium maintained the polygonal epithelial morphology of the hepatocytes and increased longevity such that over 80% of the cells survived for 3 days and at least 30% for 8 or 9 days. This enhancement of survival was obtained up to 48 hr postinoculation, but the earlier the time of dexamethason supplementation the greater the effect. Removal of dexamethasone resulted in a decrease in longevity. The positive effect of dexamethasone on longevity was observed following dexamethasone replacement of insulin in supplemented cultures, but the combination of insulin and dexamethasone resulted in poorer survival than with dexamethasone alone. The results are interpreted to indicate that dexamethasone provided a requirement of the in vitro environment for survival and suggest that elaboration of a complex medium is required to maintain hepatocytes in culture.