Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the proliferation and maturation of immature myeloid progenitor cells and primes mature cell function in phagocytes. To investigate whether the biochemical events following the binding of GM-CSF to its receptor are differentiation dependent we analysed GM-CSF mediated activation of the JAK 2-STAT 5 and MAP kinase pathways in undifferentiated HL-60 cells and HL-60 cells induced to differentiate with dimethyl sulphoxide (DMSO) or retinoic acid (RA). GM-CSF stimulated MAP kinase activation in both the undifferentiated and differentiated HL-60 cells. Activation of MAP kinase (expressed as a proportion of total cellular MAP kinase) was maximal at 5 min and of similar magnitude in both cell types. There was, however, a marked difference in the later kinetics of activation, with the response being transient in the undifferentiated cells and disappearing within 15 min, whereas it was prolonged and persisted for at least 60 min in the differentiated cells. GM-CSF mediated activation of STAT 5 was markedly increased (15-20-fold) after differentiation of HL-60 cells but the kinetics of activation did not change. The increase in STAT 5 activation was not due to a change in total cellular STAT 5 expression but correlated with increased JAK-2 protein levels. These data show that in the HL-60 cell model, differentiation modulates the activation of signalling molecules downstream of the GM-CSF receptor.