The transformation of certain cells reduces the requirement of extracellular Ca'+ for growth. The SV-40 transformed human lung fibroblasts, WI-38 VA13, require less Ca" than normal WI-38 cells. Spreading area of the normal cells decreases when cultured in 1 0 p M Ca2+ medium. lntracellular calcium concentration ([Ca2+],) of the normal and transformed cells cultured in 10 p, M and 2 m M CaL+ media was measured by the fluorescence microscope technique using fura-2 as a probe. The [Ca2+], is measured in the resting state and during mobilization by serum or bradykinin stimulation. The lowering of extracellular calcium concentration results in a decrease in the resting state [Ca'+], of both normal and transformed cells. Although the total decrease in ICa'+], is the same for both cells, the rate of decrease is much faster in normal cells than in transformed cells. Low extracellular Ca'+ reduces the number of cells responsive to the serum or bradykinin stimulation and decrease5 the peak [Ca"li value in both cells. In addition, we investigated, using BCECF as a fluorescent probe, the intracellular p H (pH,) of normal and transformed cells maintained at low and normal Ca2 ' . The low CaL+ condition makes pH, acidic in normal cells but not in transformed cells. The acidification of'the normal cell is accompanied by a decrease in the spreading area of the cells. The decrease of the cell attachment, followed by the reduced spreading area, induces the acidic pH,. These results suggest that the reduced Ca' ' requirement of transformed cells for growth is related to the mechanism of pH, regulation rather than Ca2+ homeostasis and, possibly, to the anchorageindependent growth, which is a unique feature of transformed cells.