The T cell-derived lymphokines interleukin 2 (IL-2) and interleukin 4 (IL-4, originally BSF-1) both exhibit T cell growth-promoting activity. Recent observations that T cell lines commonly used as indicator cells in IL-2 bioassays also proliferate in response to IL-4 demonstrate the lack of specificity of these bioassays for IL-2. In this report we describe a bioassay method to differentiate IL-2 and IL-4 through the parallel use of two T cell lines with defined lymphokine specificity. The IL-2-responsive CT6 cell line, when used in conjunction with the IL-2- and IL-4-responsive HT-2 cell line, allows for the differentiation of IL-2 and IL-4 in conditioned media.