The microstructure of human tumor necrosis factor alpha (TNF-a) and its mutant (TNF-b) has been investigated by utilizing positron annihilation lifetime spectroscopy, radioiodination of human TNF and L929 cells assay. The experimental results show that the long lifetime (T₂) and corresponding intensity (I₂) of lower ortho-positronium annihilation in TNF-a are longer and less than those in the TNF-b, respectively. It suggests that the TNF-b is smaller in free volume and higher in density than the TNF-a. The TNF-b may maintain a more favorable conformation for binding to TNF receptors, thus increasing its biological activity. It is then concluded that the increases in the cytotoxicity and in the density for the TNF-b result from the decreases in the free volume in the TNF-b.