IL-3 is a recently described lymphokine that induces the expression of the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in an early T-cell precursor. We demonstrate that purified IL-3 an be used to establish continuous cultures of a discrete subpopulation of T cells with virtually 100% efficiency from normal, unstimulated splenic lymphocyte populations. Once established over a period of approximately 4-6 weeks, the cultures can be readily cloned either in soft agar or by limiting dilution. All of the established lines are Lyt1+, 2- la-, lg-, Tdt-, 20 alpha SDH+, a phenotype characteristic of helper T cells; they are therefore distinct from continuous cultures of T cells established with IL-2. although initiation of these cell lines was absolutely dependent on IL-3, once established all of the cell lines were independent of exogenously added lymphokines for their growth in vitro. However, all of the cells lines were found to constitutively produce IL-3 at high levels. None of the cell lines constitutively produced lL-2, but could be readily induced to produce this lymphokine by treatment with phorbol-myristic acetate. The ability to produce lL-3 and lL-2 is a further indication that all the cell lines are helper T cells. The possible mechanisms by which lL-3 allows the specific differentiation and/or amplification of T cells of helper phenotype in tissue culture are discussed.