Three assays of murine IFN gamma are compared in terms of sensitivity, intra- and inter-assay variability, specificity and simplicity. The widely used viral inhibition assay requires 48 hours, necessitates continuous maintenance and optimization of fibroblast growth, and exhibits the lowest sensitivity. Inhibition of WEHI-279 B cell [3H]thymidine incorporation requires 48-60 hours to quantitate IFN gamma production, can be subject to non-specific inhibition, and is also labor intensive. In both bioassays, specificity must be determined by the analysis of duplicate samples in the presence of neutralizing, IFN gamma-specific mAb. In contrast, a 24 hour, dual mAb ELISA, in which IFN gamma is captured by immobilized, purified rat IgG1 XMG 1.2 mAb and identified with biotinylated mAb R4-6A2 and streptavidin-alkaline phosphatase detects IFN gamma production > 0.05 U/ml. The quantitative range in this assay is typically from 1-100 U/ml. In addition to providing the greatest specificity and shortest duration, this ELISA exhibits the lowest coefficient of variation of the three assays compared. Collectively, assay characteristics such as sensitivity, absence of interference by other proteins, reproducibility, speed and simplicity support the conclusion that this dual mAb based sandwich ELISA represents a substantial improvement over inhibition of viral cytopathic effect or inhibition of WEHI-279 bioassays for characterization of antigen- or mitogen-driven IFN gamma production.