A spontaneously transformed clone was isolated from repeatedly passaged BALB/c 3T3 cells. The transformed cells were rounded or slender and elongated, were randomly arranged in an overlapping pattern, grew to high cell density, and had a low requirement for serum. The rates of multiplication and DNA synthesis of the nontransformed and the transformed lines were reduced for several days by drastic reduction in the Mg2+ concentration of the medium, but the rate of DNA synthesis in the Mg2+-deprived cultured increased after 6--8 days, suggesting an adaptation of the cells or a change in local environment. When maintained in very low Mg2+ concentrations the transformed cells assumed the appearance and arrangement of nontransformed cells within 1 day. The rate of DNA synthesis in the transformed cultures in 1.0 mM Mg2+ was independent of serum concentration. After 3 days of Mg2+ deprivation, however, the rate of DNA synthesis became highly dependent on both serum concentration and population density, thus resembling the growth behavior of nontransformed cells. Neither deprivation of K+ or Ca2+ nor addition of dibutyryl cyclic AMP produced these effects. The Mg2+ contents of nontransformed and transformed cells in physiological concentrations of Mg2+ were similar and only slightly reduced by incubation for 4 days in Mg2+-deficient medium. In 1.0 mM Mg2+, the Ca2+ content of the nontransformed cells was approximately 3 times higher than that of the transformed cells. After incubation in Mg2+-deficient medium, the Ca2+ contents of both cells types increased; that of the transformed cells slightly exceeded that of the nontransformed cells in Mg2+-sufficient medium. The results show that Mg2+-deprived transformed cells closely resemble nontransformed cells in appearance, requirement for serum, response to cell population density, and Ca2+ content. They also show that these parameters can be regulated coordinately by Mg2+ and support the suggestion that a defect in regulation by Mg2+ is a basic feature of the malignant transformation.