Details are presented of a technique for maintaining adult liver parenchymal cells in culture in a nonproliferating state. Hepatic regenerative stimulator substance (SS), prepared from weanling rat liver, will stimulate tritiated thymidine incorporation into liver DNA when injected into another rat or mouse. Preliminary experiments are presented which show that SS stimulates non-proliferating adult hepatocytes to incorporate increased amounts of (3)HT dr into DNA when SS is added to the culture medium. The response is dose dependent and a lag period of about 12 hours exists between addition of SS to the cultures and the stimulation of DNA synthesis. Preliminary screening of several cell lines in culture reveals that only liver related cell lines (hepatomas) respond to SS. A brief review of other hepatotrophic factors is also included.