Immortalized CWSV-1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, had increased numbers of cells with strand breaks in genomic DNA (terminal dUTP end labeling) when grown in 0 μM choline (67-73% of cells) than when grown in 70 μM choline (2-3% of cells). Internucleosomal fragmentation of DNA (DNA ladders) was detected in cells grown with 5 μM and 0 μM choline for 72 h. Cells treated with 0 or 5 μM choline for 72 h detached from the substrate in high numbers (58% of choline deficient cells vs. 1.4% of choline sufficient cells detached) exhibited a high incidence of apoptosis (apoptotic bodies were seen in 55-75% of cells; 67-73% had DNA strand breaks), and an absence of mitosis and proliferating cell nuclear antigen (PCNA) expression. Cells undergoing DNA fragmentation had functioning mitochondria. At 24 h, cells grown in 0 or 5 μM choline synthesized DNA more rapidly than those grown in 70 μM choline. By 72 h, the cells grown in 0 or 5 μM choline were forming DNA much more slowly than control cells (assessed by thymidine incorporation, PCNA expression, and mitotic index). Western blot analysis showed that p53 in the nucleus of cells was detected in direct association with SV40 T-antigen, and was therefore likely to be inactive. We conclude that choline deficiency kills CWSV-1 hepatocytes in culture by inducing apoptosis via what may be a p53-independent process, and that this process begins in viable cells before they detach from the culture dish.—Albright, C. D., Liu, R., Bethea, T. C., da Costa, K.-A., Salganik, R. I., Zeisel, S. H. Choline deficiency induces apoptosis in SV40-im- mortalized CWSV-1 rat hepatocytes in culture. FASEBJ. 10,510-516(1996)