Vesicular stomatitis virus (VSV), a mammalian RNA virus enclosed by a lipid bilayer membrane of host cell origin, is inactivated by normal human serum. Several possible mechanisms of this inactivation were examined. 1) Neutralization by antibody alone. This possibility was ruled out by showing that virtually all (>90%) of the neutralizing activity was lost after heating the sera. Also most preparations of human IgG, IgA, or IgM failed to neutralize VSV. 2) Neutralization by a combination of low levels of antibody together with complement. Anticellular antibody is unlikely since human sera effectively neutralized VSV grown in cell lines from multiple species even after absorption with uninfected cells in which the virus had been grown. Similarly, anti-viral antibody appears unlikely since normal human serum effectively neutralized after absorption with cells expressing VSV antigens. Furthermore, purified human IgG did not potentiate the neutralizing ability of normal serum.