Technics of double diffusion in tubes with an agar gel have been applied to the study of various pox-virus antigens. The diffusion pattern of vaccinia antigen against standard vaccinia serum is composed of 3 to 6 zones of specific precipitation on different levels. Two of the corresponding fractions (I and III), which are distinctly precipitated, should be considered as major fractions. The other, more faintly precipitated minor fractions, were not always present (2, 2′, 4 and 5). Elementary

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... ies play no role in this precipitation. There is no essential difference between the patterns of vaccinia, neurovaccinia, rabbit-pox, smallpox and alastrim diffusing against standard vaccinia serum. Only the minor fraction 2 of a vaccinia suspension could be distinguished from the minor fraction 2′ of an alastrim suspension by mixing the suspensions of the two strains (differential test). As higher concentrated vaccinia suspensions may show both 2 and 2′ fractions, this is not a point of differentiation. The major precipitation zone I is lacking in the diffusion patterns of the seven cowpox strains and the ectromelia strain. Cross tests, however, show that cowpox possesses a fraction (I) with the same diffusion properties as fraction I of vaccinia, but distinguished by a more limited reactivity in vitro; whereas fraction I of vaccinia is precipitated by vaccinia and cowpox serum, fraction (I) of cowpox is only faintly precipitable with cowpox serum and not at all with vaccinia serum. A simple serological method of differentiation between cowpox and vaccinia based on this point of distinction has been described.