The biological significance of the existence of multiple interferon-alpha (IFN-alpha) subtypes is unknown but may represent a finely tuned mechanism whereby different subtypes are produced in response to different stimuli. To investigate the expression of individual IFN-alpha subtypes, polyclonal antipeptide antisera designed to react with all IFN-alpha subtypes, or with a particular subtype, IFN-alpha 2 or IFN-alpha 4, have been produced. In this study we demonstrate the utility of these antisera for the detection, using indirect immunofluorescence staining, of intracellular IFN-alpha produced by human peripheral blood mononuclear cells (PBMC) and lymphoblastoid cells. Secreted IFN-alpha was also investigated by bioassay and a sandwich radioimmunoassay (RIA), using two monoclonal antibodies (mAb) and specific for IFN-alpha 4. The PBMC were shown to produce IFN reactive with all three polyclonal antisera, after stimulation with Sendai virus. The lymphoblastoid cells also produced IFN, including IFN-alpha 2, but IFN-alpha 4 was not detected either intracellularly, by immunofluorescence, or in the medium, by sandwich RIA. The immunofluorescence studies also demonstrate that in the absence of viral stimulation IFN-alpha is found in the cytoplasm of PBMC and lymphoblastoid cells but not secreted in detectable levels. The finding that two lymphoblastoid cell lines do not produce the subtype IFN-alpha 4 raises important questions as to whether other cell lines and cell types produce IFN-alpha subtypes selectively, and whether individual IFN-alpha subtypes have different roles in human physiology and pathology.