The influence of four liver tumor promoters and two structurally related chemicals (non-promoters) on survival of normal adult rat hepatocytes was examined in primary culture. Of the four promoters, butylated hydroxytoluene (BHT), dichlorodiphenyltrichloroethane (DDT), barbital sodium and phenobarbital sodium, only phenobarbital efficiently prolonged the hepatocyte survival and maintained the morphological features of the cells. Both BHT and DDT were toxic to hepatocytes within the dose ranges tested. Barbital was also ineffective for maintenance of primary cultured hepatocytes but not toxic to the cells. Of the two non-promoters, barbituric acid and amobarbital, barbituric acid also showed no maintenance effect on hepatocytes. However, amobarbital resembled phenobarbital in its effect on the maintenance of hepatocytes in primary culture. DNA synthesis of primary cultured hepatocytes was severely suppressed by phenobarbital in a dose-dependent manner. The results clearly show that the ability to support long-term survival of primary cultured hepatocytes is a common property of some barbiturates but not of liver tumor promoters, and that the maintenance of hepatocytes by phenobarbital is not due to a counterbalance of stimulated proliferation and death of the cells.