Analysis of chemically cross-linked recombinant human tumor necrosis factor (rTNF) on sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the presence of three distinct molecular forms which correspond to trimers, dimers, and monomers. The cross-linking procedure appeared to enhance the antigenicity of these molecular forms of rTNF as determined by Western blot analysis. Cross-linking was unaffected by 2% Nonidet P-40, high concentrations of human serum albumin, or 60% normal human serum. Sodium dodecyl sulfate (0.08%) completely abolished the cross-linking of rTNF. This paper demonstrates that rTNF, at a concentration as low as 0.8 ng/ml, exists solely in the trimeric form in solution under nondenaturing conditions.