The level of unscheduled DNA synthesis in the parenchymal cells from hyperplastic nodules and from the entire liver of rats fed N-2-fluorenylacetamide was studied and compared with that of normal liver cells. Measurements of unscheduled DNA synthesis were carried out by the use of a primary liver cell culture system. Livers were perfused with collagenase, the cells from individual hyperplastic nodules, and/or from the whole liver aspirated and plated onto plastic Petri dishes. Simultaneous histochemical measurements of beta-glucuronidase were carried out in the cultured cells as an aid in distinguishing functional cell types. The cells from hyperplastic nodules obtained from the liver during carcinogen feeding survived much longer than normal liver cells in culture. The level of unscheduled DNA synthesis was determined radioautographically after exposing cells to ultraviolet light and incubating with [3H]thymidine. [3H]Thymidine labeling was variable among individual nodules or animals and fluctuated as a function of the number of days in culture. In general, however, the level of unscheduled DNA synthesis in the cells from hyperplastic nodules was always higher than or similar to that of normal liver cells. Thus, the cells of hyperplastic nodules are not more readily transformed into the malignant state than normal cells as a result of their lowered DNA repair mechanisms.