Herpes simplex virus type 1 (HSV-1) encodes a protease that is essential for proteolytic processing of itself and of the nucleocapsid-associated protein, ICP35 (infected cell protein 35) (Liu, F., and Roizman, B. (1991) J. Virol. 65, 5149-5156). Inhibitor studies indicated that the HSV-1 protease is sensitive to the serine protease inactivator diisopropyl fluorophosphate (DFP). Inactivation is irreversible and dependent on time and concentration of DFP. Loss of activity correlates linearly with the incorporation of [3H]DFP. Analysis of completely inactivated protease by mass spectrometry indicated a stoichiometry of 1 DFP/protease. In order to identify the specific residue modified by DFP, the protease was labeled with [3H]DFP and subsequently digested with trypsin or chymotrypsin. The peptides resulting from each digestion were separated by reverse phase HPLC, and the radioactivity was recovered in a single peak. Mass spectrometric studies and sequencing analysis by Edman degradation identified Ser-129 as the residue modified by DFP. This residue and the region in which it is found is highly conserved among the herpes viral proteases. These data demonstrate that HSV-1 protease is a serine protease and that Ser-129 is the active site nucleophile.