Although several sensitive and specific assays have been developed to quantify murine cytokines, these assays do not allow individual cells to be correlated with the specific cytokines they produce. The purpose of this study was to develop a sensitive and reproducible method for the detection of individual T cells which secrete either interferon-gamma (IFN-gamma) or interleukin-5 (IL-5). We have used an adaptation of the enzyme-linked immunospot (ELISPOT) assay in which monoclonal antibodies to IFN-gamma (R4-6A2) and to IL-5 (TRFK-5) were used to coat 96-well plates with a nitrocellulose base. Mouse splenic T cells, either nonstimulated or activated with concanavalin A (ConA) or phytohemagglutinin (PHA), were cultured in individual wells. Following incubation, the cells were removed, and the bound cytokines probed with either biotinylated mAb anti-IFN-gamma (XMG 1.2) or anti-IL-5 (TRFK-4) followed by avidin-peroxidase. The spots which developed with 3-amino-9-ethylcarbazole were discrete and enumerated with a dissecting microscope. Although unstimulated splenic T cells contained low numbers of cytokine-specific spot-forming cells (SFC), 24-72 h activation with mitogen was required to induce significant numbers of cytokine producing cells. When mitogen-stimulated splenic CD4+ T cells were assessed, approximately equal numbers of IFN-gamma and IL-5 SFC were seen. Approximately 20-30% of all mitogen-activated splenic T cells produced at least one of these two cytokines. Pre-incubation of biotinylated anti-IFN-gamma with recombinant IFN-gamma (rIFN-gamma) or anti-IL-5 mAbs with rIL-5 completely inhibited cytokine-specific SFC. Further, use of nonrelevant antibodies did not result in spot formation, and treatment of mitogen-activated T cells with cycloheximide inhibited both IFN-gamma- and IL-5-specific SFC. A sensitive method has been developed which allows detection of individual T cells that produce either IFN-gamm or IL-5, and should be useful for detection of cytokine secretion at the single cell level.