RNA-RNA in situ hybridization was used to study the frequency of cells producing mRNA for IL-2 and IL-2-R in T lymphocytes stimulated by either of three mitogens: anti-CD3, anti-CD28, or PHA. Both CD4+ and CD8+ T cells expressed transcripts for IL-2 and the low-affinity IL-2-R when stimulated with these mitogens plus PMA. IL-2 transcripts peaked at 8-16 h, and IL-2-R at 24-40 h. Cyclosporin A (CSA) inhibited the synthesis of IL-2, but not IL-2-R mRNA, after stimulation by PHA or anti-CD3. However, higher concentrations of CSA were necessary to achieve 50-70% inhibition after stimulation with anti-CD28. At optimal points 12-22% of CD4+ and 5-13% of CD8+ cells expressed IL-2 mRNA, while 30-50% of cells of both subsets had IL-2-R mRNA. The IL-2 grain counts, which relate to the level of mRNA/cell, were higher in the CD4+ subset but could be increased several fold in the CD8+ subset in the presence of adherent accessory cells. The use of PMA as an accessory stimulus, in addition to adherent cells, greatly increased the frequency of lymphocytes with IL-2 mRNA and the amount of IL-2 activity in the culture medium, but the proliferative response was not significantly boosted. These observations indicate at the single cell level that many CD4+ or CD8+ lymphocytes can make IL-2 mRNA, and that the induction of IL-2 with several stimuli is reduced by CSA and enhanced by PMA.