The transformation of certain cells reduces the requirement of extracellular Ca2+ for growth. The SV-40 transformed human lung fibroblasts, WI-38 VA13, require less Ca2+ than normal WI-38 cells. Spreading area of the normal cells decreases when cultured in 10 microM Ca2+ medium. Intracellular calcium concentration ([Ca2+]i) of the normal and transformed cells cultured in 10 microM and 2 mM Ca2+ media was measured by the fluorescence microscope technique using fura-2 as a probe. The [Ca2+]i 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 [Ca2+]i of both normal and transformed cells. Although the total decrease in [Ca2+]i is the same for both cells, the rate of decrease is much faster in normal cells than in transformed cells. Low extracellular Ca2+ reduces the number of cells responsive to the serum or bradykinin stimulation and decreases the peak [Ca2+]i value in both cells. In addition, we investigated, using BCECF as a fluorescent probe, the intracellular pH (pHi) of normal and transformed cells maintained at low and normal Ca2+. The low Ca2+ condition makes pHi 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 pHi. These results suggest that the reduced Ca2+ requirement of transformed cells for growth is related to the mechanism of pHi regulation rather than Ca2+ homeostasis and, possibly, to the anchorage-independent growth, which is a unique feature of transformed cells.