A filter immuno-plaque assay was developed which detects alpha interferon (INF-alpha)-secreting human peripheral blood leucocytes (PBL). Polyclonal anti-IFN-alpha antibodies were fixed to the nitrocellulose membrane bottoms of 96-well Millititer plates, which also contained monolayers of glutaraldehyde-fixed human WISH amnion cells infected by Herpes simplex virus type 1 (HSV). Such cells are potent IFN inducers and during a 16 h cocultivation with PBL, IFN-alpha was absorbed by the membrane-bound polyclonal antibodies around IFN-alpha-secreting cells. This IFN-alpha was detected with murine monoclonal antibodies against IFN-alpha and peroxidase-labelled antibodies against murine immunoglobulin, using diaminobenzidine as substrate. Distinctly stained plaques were seen, the frequency of which gave a minimal estimate of approximately 10 IFN-alpha-producing cells in 10(4) PBL (range in 12 blood donors 2.95-25.1). Fewer plaques than expected were seen at low PBL numbers per culture, one explanation being that cell interactions then limit the IFN-alpha response. The immuno-plaque assay should be useful in further studies of the cellular basis of the IFN-alpha response.