This report was presented to the Subcommittee on Fat-Soluble Vitamins of the WHO Expert Committee on Biological Standardization in 1949 by the Vitamin D Sub-Committee of the Accessory Food Factors Committee of the Medical Research Council of Great Britain and formed the evidence on which the first-mentioned subcommittee based its recommendation of the adoption of crystalline vitamin D(3) as the International Standard for Vitamin D, replacing the solution of irradiated ergosterol which had been adopted as Standard in 1931.The Vitamin D Sub-Committee organized a collaborative assay in which 32 laboratories in seven countries participated. In addition to 29g of crystalline vitamin D(3) generously contributed by five firms, the following preparations were compared for vitamin D activity by biological tests on rats or chicks or both: the then current International Standard, a preparation of the purest calciferol obtainable, the British Standards Institution (BSI) Standard, the United States Pharmacopeia (USP) Reference Cod Liver Oil, and the Swedish provisional standard.Altogether 29 rat assays and 25 chick assays were carried out, and the results were subjected to standard methods of analysis. The BSI Standard and the pure calciferol were found less potent than the Old Standard (potency ratios: 0.916 and 0.933, respectively); and the New Standard may be slightly less potent (0.981), but it is more potent than the BSI Standard (1.071). The USP Reference Oil is less potent than the BSI Standard (0.949), the Old Standard (0.896), and the New Standard (0.886). The fiducial range is generally less than 10%, but even where it is greater, it is still satisfactory for a biological test.