In a collaborative assay initiated by the International Association of Biological Standardization 13 laboratories investigated the properties of 10 freeze-dried BCG vaccines made in different ways by different producers which met the requirements of the World Health Organization. The in vitro tests with respect to the dry weight of the bacteria, the number of culturable particles and the stability during storage at 37 degrees C gave results which showed statistically significant differences between vaccines as well as between laboratories. The use of a reference vaccine in the tests for the number of culturable particles did not improve the precision of the results. These tests are useful as in-process tests to assure the producer that his vaccine will consist of the required amount of bacteria of which a high percentage is living. It was found that all vaccines had a survival of more than 25% after 1 month at 37 degrees C with the exception of two vaccines in rubber-stoppered vials. The two in vivo tests, determining skin reactivity in guinea-pigs and multiplication of BCG in spleens of mice, resulted in nearly the same ranking of the vaccines with respect to their virulence, differing from that of the in vitro tests. In the skin reactivity test the simultaneous injection of both the reference vaccine and a test vaccine into the same animal achieved the elimination of the inter-laboratory variation of the results.