Changes in the expression of two isoenzymic forms of pyruvate kinase in fetal hepatocyte cultures derived from 15- and 19-day gestation rats are studied by immunocytochemical localization of the respective antigens. Initially, in cultures established from 15-day gestation rats only the 'embryonic' form of the enzyme (M2-PK) is detected in all cells. Cells which stain positively for the liver specific form of the enzyme (L-PK) are not observed. After 2 days' culture, a significant number of cells have become positive for L-PK. All the positive cells have a morphology which is typical of liver parenchymal cells. However, the majority of parenchymal cells remain negative for L-PK while retaining M2-PK. In contrast, all cells which display a fibroblastic morphology, as well as clear epithelial cells are M2-PK positive, but L-PK negative. In 5-day-old cultures, all hepatocytes have become L-PK positive. Hepatocytes derived from 19-day gestation rat liver stain positively for L-PK on day 1 of culture in agreement with previously published biochemical data. A minor population of negative cells is non-parenchymal in appearance. All parenchymal cells are negative when the culture is stained with M2-PK specific antibody. Five days after the culture is established, many non-parenchymal cells are present. Such cells are L-PK negative and M2-PK positive and their presence in cultures derived from both 15- and 19-day gestation rats explains the persistence of M2-PK. This study reveals that during enzymic differentiation of fetal hepatocytes, all immature hepatocytes are initially capable of expressing M2-PK while they do not produce L-PK. During culture, a sub-population of these cells initiates synthesis of L-PK, indicating that only a fraction of the cells differentiate. At the same time, hepatocytes which do not stain for M2-PK appear, which suggests that cells which initiate L-PK synthesis have ceased to make M2-PK. Eventually all hepatocytes are L-PK positive and M2-PK negative, indicating that a switchover in expression of the pyruvate kinase isoenzymes has occurred.