1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dependent growth and differentiation of 6 tumor cell lines has been determined by the use of the monolayer proliferation assay. Cell lines of 4 gastro-intestinal carcinomas, 1 malignant schwannoma, and 1 malignant histiocytoma have been established and characterized. Cells were incubated for 4, 7, and 11 days in the presence of 0.8 or 8 nM 1,25(OH)2D3 and for control without addition of the hormone. Proliferation rates of 1,25(OH)2D3 treated cells were compared with cell growth in the untreated controls. Five out of 6 cell lines showed a 1,25(OH)2D3 dependent growth pattern. With 8 nM 1,25(OH)2D3 they were all inhibited. With 0.8 nM, 3 of them were inhibited at any time of the test period, whereas 1 was stimulated at day 4 and inhibited at days 7 and 11. One cell line was stimulated at days 4, 7, and 11 when incubated with 0.8 nM 1,25(OH)2D3. No striking morphological changes could be observed in the presence of 1,25(OH)2D3. We conclude that 1,25(OH)2D3 dependent cells in vitro are not necessarily growth-inhibited by this compound. Thus, 1,25(OH)2D3 is not an exclusively proliferation inhibiting agent.