The infectivity of vesicular stomatitis virus (VSV) was moderately affected by iodoacetic acid and drastically affected by N-ethylmaleimide; the antiviral effect of these sulihydryl reagents was enhanced somewhat by the reducing agent, 2-mercaptoethanol. Reducing and/or a!kylating reagents did not affect VSV hemagglutination and the impermeable sulfhydryl reagent, dextran-maleimide, did not significantly influence VSV infectivity. These data indicate that glycoprotein spikes are not the major sites for the antiviral activity of sulfhydryl reagents. ["C]Iodoacetic acid was able to penetrate the virion membrane to bind covalently to the free sulfhydryl groups of all five virion proteins, particularly the reduced disulfides of the L protein. The RNA polymerase activity of intact VSV was inhibited by iodoacetic acid and to a greater extent by N-ethylmaleimide, which probably accounts for the loss of viral infectivity caused by the permeable sulfhydryl reagents.