We have previously described the use of a chemically defined medium (CDM) supplemented with epidermal growth factor (EGF) and dimethylsulfoxide (DMSO) to maintain long‐term cultures of rat hepatocytes in a highly differentiated state. In this study, conditions necessary to stimulate high levels of DNA synthesis in hepatocytes in long‐term DMSO culture were defined. Hepatocytes were maintained in culture for 20 days in CDM containing DMSO and EGF, insulin, and glucagon. EGF, insulin, and glucagon were then removed for 7 days. Readdition of EGF, insulin, and glucagon at day 27 (shiftup) was accompanied by a three‐ to sixfold increase in labeling index. If DMSO or dexamethasone (dex) + DMSO were removed at time of shiftup, the labeling index increased by 18‐ to 54‐fold. TGFβ inhibited DNA synthesis stimulated by EGF shiftup, TGFα shiftup, or EGF shiftup in combination with removal of dex + DMSO. Stimulation of DNA synthesis was accompanied by a specific, sequential induction of protooncogene mRNA levels; c‐fos mRNA was induced 23‐fold at 0.5 h after readdition of EGF; c‐myc mRNA was induced three‐ to fourfold by 0.5 h; TGFα mRNA was induced sevenfold by 8 h; K‐ras mRNA was induced fourfold by 26 h. Changes in protooncogene expression paralleled changes seen in regenerating liver. When DMSO was removed for greater than 48 h, the cells flattened and spread out, chords of cells were no longer well defined, albumin mRNA levels decreased, and fibronectin, β1 integrin, and TGFβ transcripts increased. © 1993 Wiley‐Liss, Inc. Copyright © 1993 Wiley‐Liss, Inc.