In 1950 the Department of Biological Standards, National Institute for Medical Research, London, was authorized by the WHO Expert Committee on Biological Standardization to prepare the Second International Standard for Penicillin. A single batch of specially recrystallized sodium penicillin G was obtained and 11 laboratories in seven different countries were requested to take part in its collaborative assay. 112 assays were carried out, of which 101 were done by cup-plate methods using either Staphylococcus aureus or Bacillus subtilis. The results were subjected to standard methods of analysis, on the basis of which the authors define the Second International Standard for Penicillin as containing 1,670 International Units (IU) per mg, with limits of error (P = 0.05) of 1,666-1,674 IU/mg. The International Unit is therefore redefined as the activity contained in 0.0005988 mg of the Second International Standard for Penicillin.