A limiting dilution assay capable of detecting interleukin 4 (IL4) production by a single cell has been developed. This assay is based on the stimulation of T cells, in the presence of IL2 (5 U/ml), with anti-CD3 antibody bound to the surface of Terasaki wells. Cells of the IL4-selective indicator line, CT.4S, are added 24-36 h later and IL4 production is determined based on their survival 24-48 h thereafter. A frequency of 0.98 was obtained for IL4 production by T cells of the D10.G4 cell line. T cells from naive donors capable of producing IL4 in response to anti-CD3 plus IL2 were quite rare, with a frequency in four experiments ranging between 0.0003 and 0.0018. Treatment of mice with polyclonal activators known to increase the IL 4-producing capacity of T cells when assayed in bulk culture caused striking increases in the frequency of IL4-producing cells. Similarly, culturing cells in vitro with anti-CD3, IL2 and IL4 for 5 days caused a marked increase in the frequency of cells capable of producing IL4, to 0.031.IL4 production by individual T cells is dependent upon IL2. Thus, in naive T cell populations, the frequency of IL4-producing cells in response to stimulation with anti-CD3 in the absence of IL2 was below the limit of detection. T cells from primed donors showed a striking inhibition in the frequency of IL4-producing cells in response to anti-CD3 when IL2 was not present. The availability of a simple assay to measure the frequency of cells capable of producing IL4 should have substantial utility in allowing the evaluation of conditions that regulate IL4 production in vivo and in vitro.