To assess mice interleukin-12 (IL-12)-secreting cells at a single cell level, we have developed a murine IL-12 specific enzyme-linked immunospot (ELISPOT) assay. The application of the newly developed method clearly showed the frequency of IL-12-secreting cells in the resident peritoneal exuded cells was higher than other organs of normal DBA/1J mouse. Moreover, we determined the frequency of IL-12-secreting cells in the spleens of five strains of inbred mice, and found the incidence of IL-12 secretors in the strain C57BL/6 to be greatest, and significantly greater than four of the others. These results are compatible with the predicted evidence, supporting this ELISPOT assay for IL-12-secreting cells is accurate. The procedure provides a useful tool for investigating complicated immune responses at a single cell level.