Macrophage activation factor (MAF) and migration inhibitory factor (MIF) obtained from sensitized human lymphocytes stimulated by streptokinase-streptodornase (SK-SD) were characterized by gel filtration and isoelectric focusing (IEF) techniques. Twenty-four and 48 hr supernatants were chromatographed on Sephadex G-100 columns and the eluate pooled into 5 fractions: the void volume (Fr. I), the eluate containing molecules with a molecular weight ranging from 55-70,000 (Fr. II), 30-55,000 (Fr. III), 20-30,000 (Fr. IV) and 10-20,000 (Fr. V). The 24 hr supernatants contained MIF activity in Fraction IV and maximal MAF activity in Fractions I and II whereas the 48 hr supernatants contained MIF activity in Fractions II and IV and maximal MAF activity in Fractions II and III. When the supernatants were purified by IEF, the eluant was pooled into 5 fractions: Fraction I (pI 3.5-4), Fraction II (pI 4-4.5), Fraction III (pI 4.5-5), Fraction IV (pI 5-5.5) and Fraction V (pI 5.5-6). The 24 hr supernatants contained maximal MIF activity in Fractions III and IV and maximal MAF activity in Fraction III whereas the 48 hr supernatants contained both maximal MIF and MAF activity in Fractions I and IV. Thus, it appears that although human MAF can be differentiated from MIF in the 24 hr supernatants, MAF and MIF activity in the 48 hr supernatants are generally found in the same fractions when examined either by IEF or gel filtration techniques.