Characterization of the heat-labile variola virus inhibitor (HLI) present in normal hu man and guinea pig serum (GPS) was attempted. The HLI activity was destroyed by 2-mercaptoethanol, acetone, dialysis against acid and UV-irradiation, but was resistant to oxidation with KIO4. Sucrose density gradient centrifugation of GPS located the HLI activity in fractions sedimenting faster than IgM, whereas the complement activity was found in the slower sedimenting IgG + IgA region. The serum fraction containing HLI, obtained by sucrose density gradient centrifugation, did not enhance neutralization by an early antiserum, whereas the complement fraction did. The HLI activity of the above isolated fraction was far more sensitive to trypsin than that of the whole serum. Infectivity of HLI-neutralized variola virions was restored by short digestion with 0.01 % trypsin, while sonication had no such effect. Neutralization with HLI did not inhibit virus adsorption to cells but blocked the induction of early soluble antigen (s-antigen).