A limiting dilution procedure using lymphocytes isolated by FACS is described to assess the role of individual T cell subsets for the production of IL-2 in the presence of purified B (Leu-12+) cells. Purified B cells were isolated by two-color FACS using B (anti-Leu-12) and T (anti-Leu-4) cell specific fluorescent conjugated antibodies. The Leu-12+ cells were treated with anti-Leu-5b plus complement to remove contaminating sources of IL-2 producing cells and titrated with purified T cell subsets using a miniassay to determine IL-2 production. Results indicated that less than 1000 Leu-4+ T cells in the presence of 98% Leu-12+ B cells could produce an equivalent amount of IL-2 produced by the 40,000 T cells estimated in the unsorted population. A comparison of the Leu-2+ and Leu-3+ T cell subsets isolated by two-color cell sorting and activated with phytohemagglutinin (PHA) demonstrated that the Leu-3+ cell population produced significantly higher higher levels of IL-2 on a per cell basis than Leu-2+ cells. Furthermore while purified preparations of Leu-3+ cells were capable of secreting small amounts of IL-2 in the absence of accessory cells, the Leu-2+ population appeared to be completely dependent upon these cells for production of this lymphokine.