The mixed leukocyte reaction is the only functional in vitro assay currently employed for confirmation of MHC matching between bone marrow recipients and their prospective donors and for MHC class II (HLA-Dw) typing. This assay is, however, time-consuming (6 days for human MLR), whereas for clinical purposes results are often required much earlier. In an attempt to shorten the MLR incubation period, we tested IL-2 (in human MLR) and IL-2/IL-3 (in mouse MLR) production as an indication of early stages of T cell activation. We here describe a shorter assay in which IL-2 and IL-3 secretion during MLR was assessed by adding the respective lymphokine-dependent cell lines either to the MLR supernatants or directly to the original MLR cultures, using the colorimetric (3-[4,5 Dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide) (MTT) technique or the 3H-thymidine incorporation assay. In both human and mouse MLR systems, lymphokine production peaked at 24-48 hr after culture initiation, allowing tests to be completed within 48 to 72 hr. Weak MLR responses, as detected by lymphokine production, could be considerably amplified by irradiating (250-1000 cGy) the responder cells and by adding heparin (1-10 U/ml) to the cultures. The results obtained by this novel procedure correlated with those obtained by the standard 6-day human MLR assay in over 250 combinations tested thus far, and therefore it may replace the standard MLR procedure.