We have produced monoclonal antibodies to bacterially synthesized, human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and have studied in detail the characteristics of three strongly neutralizing antibodies. The antibodies reacted with GM-CSF at high dilution (EC50 = 0.1-1.7 nM) in an indirect ELISA but did not react with murine GM-CSF or other cytokines. They also recognized glycosylated hGM-CSF produced by human lymphocytes. The antibodies were able to immunoprecipitate rhGM-CSF, but only reacted weakly with rhGM-CSF on Western blots, indicating that they recognized a conformation-dependent epitope. Cross-blocking studies showed that the three antibodies recognized overlapping epitopes. The antibodies inhibited binding of 125I-labeled rhGM-CSF to HL-60 cells at nanomolar concentrations and neutralized GM-CSF activity in two different bioassays. These antibodies thus provide a useful tool for analyzing the specificity of bioassays and for further studies of the production and function of GM-CSF in vitro and in vivo.