Addition of 0.1-0.5 mM sodium PP(i) for 17 hr to confluent cultures of BALB/c 3T3 cells in low serum concentrations stimulated the incorporation of [(3)H]thymidine into DNA to an extent equal to that produced by high serum concentration. PP(i) prevented much but not all of the cell detachment that accompanies decreasing the serum concentration of confluent cultures and it increased the saturation density of cultures in high serum concentrations. The stimulation had a sharp concentration dependence and was associated with the appearance in the medium of a flocculent precipitate. Stimulation and precipitate formation were dependent on Ca(2+) and inorganic orthophosphate (HPO(4) (2-)) and were inhibited by Mg(2+). More than half the Ca(2+) requirement could be met with Sr(2+). In the absence of PP(i), supranormal concentrations of either Ca(2+) or HPO(4) (2-) caused graded increases in [(3)H]thymidine incorporation and total cell yield. The effect of supranormal [Ca(2+)] depended on [HPO(4) (2-)] and vice versa, and the Ca(2+) requirement could be partially met by Sr(2+). The stimulation was associated with increasing turbidity of the medium. Various other complexing agents of Ca(2+), including the divalent cation ionophore A 23187, failed to produce stimulation of 3T3 cells. We conclude that water insoluble complexes of PP(i), HPO(4) (2-), and Ca(2+) or, at much higher concentrations, the latter two together, stimulate 3T3 cells and we speculate that this is brought about by the association of these complexes with the cell membrane.