It has been recently demonstrated that conditioned media from Con A-stimulated splenocyte cultures contain a novel lymphokine termed IL 3. IL 3 is characterized by its ability to induce 20-alpha-hydroxysteroid dehydrogenase in spleen cells from young nu/nu mice. In search of a convenient source for biochemical and in vivo studies of this lymphokine, a number of established murine cell lines were screened for the constitutive and induced production of Il 3. It was found that WEHI-3 cells, originally designated as a myelomonocyte cell line, produced high levels of IL 3 constitutively. Added mitogen and/or phorbol acetate did not enhance the production of IL 3. Production of IL 3 varied among various sublines of WEHI-3. IL 3 purified from WEHI-3-conditioned media has biochemical and biologic characteristics identical to those obtained from Con A-conditioned media. WEHI-3 conditioned media, however, generally contained 100-fold higher levels of IL 3 than conditioned media from Con A-stimulated splenic lymphocytes.