The molecular dimensions of guinea pig lymphotoxin were studied by means of gel filtration, sucrose density gradient ultracentrifugation, and CsCl isopycnic ultracentrifugation. Lymphotoxin was produced by stimulating immune lymph node cells with the specific antigen, ovalbumin, and was assayed by means of a Coulter counter microassay. The D20,w of guinea pig lymphotoxin was estimated by gel filtration on Sephadex G-100 to be (8.0 ± 0.2) × 10-7 cm2/sec, and the apparent S20,w of guinea pig lymphotoxin was estimated to be 4.2 ± 0.1S by sucrose density gradient ultracentrifugation. The buoyant density of guinea pig lymphotoxin was determined to be 1.302 ± 0.002 g/ml by CsCl isopycnic ultracentrifugation. From these values the molecular weight of guinea pig lymphotoxin was calculated to be 45,000, and the frictional ratio was calculated to be 1.1. No evidence was found to indicate that more than a single factor was involved in producing the cytotoxic effect attributed to lymphotoxin.