SUMMARY Factors in normal serum which enhance Semliki Forest virus neutralization by specific immune guinea-pig serum have been demonstrated in a range of normal domestic and laboratory animals. The factors affecting the in vitro assay system for accessory factors have been determined and enhancement of neutralization by normal guinea-pig serum in other virus/cell systems has been demonstrated. Acces- sory factors are denatured by various physical and chemical procedures but survive freeze-drying. The effect of ageing on accessory factors in vivo in normal and germ- free rats and rabbits and in normal calves, lambs, guinea-pigs and mice revealed marked species differences but showed no difference between normal and germ-free animals of the same species. There was no correlation between haemolytic com- plement and accessory factor levels present in a serum, except in rats, although the two complexes had several properties in common. 2II