During the first few hours of starvation, Dictyostelium discoideum amoebae excrete a macromolecule, probably a glycoprotein, which stimulates cell differentiation to aggregation competence. 3':5'-Cyclic AMP pulses, which mimic the chemotactic signal, and this factor (differentiation stimulating factor) are shown to exert a cooperative effect in inducing cell differentiation. Data suggest that the appearance of the factor determines the moment amoebae become responsive to cyclic AMP pulses.