Human, murine and chicken c-ets-1 proteins migrate in SDS-polyacrylamide gels as multiple species. We show here that most if not all of this heterogeneity is due to phosphorylation events occurring predominantly on serine and to a lesser extent on threonine residues. These phosphorylations can be specifically and rapidly stimulated by treatment with the calcium ionophore A23187 or abolished by lowering the extracellular calcium concentration to less than 0.1 microM. The products encoded by c-ets-2 are also phosphorylated in a Ca2+-dependent manner, indicating that these modifications have been conserved in the products encoded by different members of the same gene family. In thymocytes, where the expression of c-ets-1 is elevated as compared with other cell types, c-ets-1 protein phosphorylation occurs after stimulation with mitogenic doses of concanavalin A, is short lived and is strictly dependent upon extracellular Ca2+ sources. This suggests that the c-ets-1 gene product may play a role in the Ca2+-mediated early events linked to T-cell activation.