A mouse interleukin 3 (IL-3)-dependent cell line, IC2, could survive and proliferate over a 48-h period in the absence of IL-3 when incubated with micromolar concentrations of sodium orthovanadate. The greatest response was obtained at 12.5 microM, as judged by stimulation of cell growth. Vanadate also stimulated synthesis of nucleotides and protein in IC2 cells. After 6 h of culture in the absence of IL-3, the intracellular ATP levels of IC2 cells fell dramatically; this fall was prevented by addition of vanadate to the culture. Studies with recombinant IL-3 revealed that vanadate potentiated the effect of submaximal doses of IL-3 on the growth of IC2 cells, but did not appear to act synergistically with IL-3 to give a maximal proliferative response of the cells. After 48 h of IL-3 replacement with vanadate, IC2 cells could respond to IL-3 with no loss of proliferative integrity. These results suggest that IL-3 dependence of IC2 cells is transiently substituted for by vanadate, and it may be a useful tool to understand the mechanism of action of IL-3.