Two analogs of tumor necrosis factor-alpha (TNF-alpha) were produced by in vitro site-directed mutagenesis. In these analogs, cysteine residues at positions 69 and 101, which form a disulfide bond, were changed to alanine or leucine. CD spectra showed that the analogs are apparently similar in secondary and tertiary structure to the natural sequence TNF-alpha. In addition, the molecular size of the analogs was identical to that of the natural sequence TNF-alpha as determined by gel filtration. However, fluorescence spectra and quenching indicated that the removal of the disulfide bond alters the local conformation around tryptophan residues. The cytolytic, macrophage activation, and lipogenic activities decreased in the order of the natural sequence TNF-alpha greater than the alanine analog greater than the leucine analog, suggesting that the surface involving the disulfide bond plays a role in these biological functions and the introduced modifications decrease the activity. Differential effect of the modifications was suggested in the antiviral activity, since in this assay only the leucine analog showed significantly lower activity.