We have found that treatment of B6SUtA1 cells with 0.01% glutaraldehyde transformed them into mechanically resistant spheres, thereby making it possible to use these high interleukin 3 (IL-3) receptor-bearing cells as a solid phase reagent suitable for the large scale purification of murine IL-3 (mIL-3). Using this technique, mIL-3 was purified from serum-free pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCCM) approximately 16,000-fold using absorption to B6SUtA1 cells, Sephadex G75 superfine chromatography, and reverse phase high performance liquid chromatography on a C18 column. The overall yield was 16%. The final product consisted of two proteins with molecular weights of 19.5 and 16.5 kd. Both species possessed mIL-3-like activity. N-glycanase treatment of the purified preparation converted all of the 19.5-kd material into the lower molecular weight species, suggesting that the two species represented different glycosylated states of mIL-3 produced by activated T cells. This was confirmed by competition studies that showed that excess pure Escherichia coli-derived recombinant mIL-3, but not granulocyte-macrophage colony-stimulating factor (GM-CSF), could prevent the binding of both species of the PWM-SCCM-derived material to B6SUtA1 cells.