The enzyme-linked immunospot (ELISPOT) assay is an efficient technique for the enumeration of single cells secreting antibodies or cytokines. To assess mice interleukin 6 (IL-6)-secreting cells at a single cell level, a highly sensitive ELISPOT assay with 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and nitroblue tetrazolium chloride (BCIP/NBT) as a gel-substrate has been developed. The IL-6 specific ELISPOT assay thus developed with an ordinary plastic ELISA plate is as efficient as the previous IL-6 specific ELISPOT assay system which uses a nitrocellulose coated plate, and is much more economic and convenient. We demonstrated with this newly devised ELISPOT assay that the ratio of IL-6 producing cells in the resident peritoneal exuded cells was highest in normal BALB/c mice, and a few IL-6 secreting cells were detected in all organs we tested, namely the spleen, bone marrow, Peyer's patch, axillary lymph node and thymus.