Lipopolysaccharide-activated murine peritoneal macrophages elaborate lymphocyte-activating factor (LAF) which is mitogenic for murine thymocytes. A method of LAF production is presented that permits the generation of a relatively homogeneous molecular species. LAF has an isoelectric point of 4.8 (range 4.7-4.9). The m.w. was determined by using several physical techniques. The apparent sedimentation coefficient (S20,w) was determined to be 2.0S by sucrose gradient ultracentrifugation. The Stokes (molecular) radius was determined by gel filtration on Sephadex G-75 to be 22 A (range 21.5 to 22.5); the calculated diffusion coefficient (D20,w) was 9.7 X 10(-7) cm2/sec (range 9.5 X 10(-7) to 9.9 X 10(-7). The buoyant density of LAF is 1.30 g/cm3 (range 1.27 to 1.33) as determined by CsCl isopycnic ultracentrifugation; the partial specific volume was estimated to be 0.72 (range 0.70 to 0.74). From these data, the m.w. was calculated to be 18,000 daltons (range 16,400 to 19,600) with the Svedberg equation. The frictional ratio was calculated to be 1.25.