Eosinophilopoietin (EPP) was previously characterized by the ability to stimulate eosinophil production in vivo, but these studies could not ascertain whether EPP had a direct effect on the bone marrow or acted indirectly by causing release of eosinophilopoietic activity by other tissues. The present studies demonstrate that EPP stimulates eosinophil growth in liquid culture of mouse bone marrow in vitro. The timing of stimulation by EPP in vivo and in vitro were parallel, with maximal eosinophil growth after 48 hr. Moreover, EPP appears similar to, and possible identical with, the eosinophil growth-stimulating substance (EO-GSF) released by antigenic stimulation of immune nonadherent spleen cells. Both EPP and EO-GSF are of low molecular weight, both produce stimulation of eosinophil growth with identical kinetics, and both produced similar dose-response curves in the liquid culture system.