Human placental conditioned medium (HPCM) contains colony-stimulating factors (CSF) required for the growth in vitro of neutrophilic granulocyte-macrophage (GM) and eosinophilic (EO) progenitor cells from human bone marrow. Fractionation of CSF in HPCM was achieved by manipulation of the elution conditions on a column of phenyl-Sepharose coumn at high ionic strength (1M ammonium sulfate), all of the CSF bound; 1 sp. of GM-CSF (.alpha.) and all of the elutable EO-CSF were eluted from the column simply by reducing the salt concentration, whereas the 2nd species of GM-CSF (.beta.) was free of EO-CSF and was eluted only by increasing the concentration of ethylene glycol in the elution buffer. The 2 GM-CSF were functionally distinct. GM-CSF.alpha. preferentially stimulated colony formation by day 14 of culture, and there was a decreased proportion of neutrophil colonies and increased proportion of macrophage colonies as the strength of the stimulus was decreased; GM-CSF.beta. preferentially stimulated colony formation by day 7 of culture, and the proportion of neutrophil colonies was high (average 80%) and independent of the concentration of GM-CSF.beta.. GM-CSF.alpha. and GM-CSF.beta. were indistinguishable on the basis of apparent molecular size on gel filtration columns (MW 30,000), charge properties on isoelectric focusing beds (isoelectric point, 4.9), and were not related to each other as a sialoglycoprotein is related to its asialo form. Adherent cell removal of the target bone marrow cells (to remove colony-stimulating cells) suggested that both GM-CSF acted directly rather than by stimulating the production of GM-CSF. Mixing and titration experiments indicated that the differences in functional specificities of the 2 GM-CSF (and the lack of EO-CSF associated with GM-CSF.beta.) were not due to the presence of specific inhibitory molecules or lower absolute levels of CSF in 1 fraction relative to the other. These 2 spp. of GM-CSF should be useful in separately enumerating subpopulations of different GM-progenitor cells in human hemopoietic disorders.