Nine years of experience in our laboratory, using more than 1500 cynomolgus monkeys in 138 tests, has shown that the new neurovirulence test (NVT) adopted by the World Health Organization (WHO) for live, oral monovalent vaccine of each poliovirus type, was a reproducible and sensitive assay likely to ensure the safety of this vaccine in humans. Our findings were the following: (1) when the test vaccine and the appropriate homotypic reference vaccine were tested in a single group of monkeys, the concurrent use of the reference vaccine considerably increased the reproducibility of the NVT; (2) in the assessment of the degree of attenuation of each lot of vaccine, the use of 12 monkeys for types 1 and 2 vaccines and 20 monkeys for type 3 vaccine (inoculated intraspinally each for reference and test vaccine) was satisfactory; (3) the virus dose used per monkey (10(5.6) to 10(6.6) pfu per monkey) was found not to be critical, i.e. the lower virus dose yielded mean lesion scores in the central nervous system of monkeys at least as high or higher than the tenfold higher virus dose; (4) the statistical analysis of our data showed that the old intrathalamic (IT) assay was considerably less sensitive than the new intraspinal (IS) assay, i.e., a test vaccine with a twofold increase in monkey neurovirulence would have a 41% chance of failing in the IT test (using 30 monkeys per vaccine), while this chance increased to 99% in the WHO IS assay (using 12 or 20 monkeys per vaccine). Since the introduction of the WHO NVT in Canada, the laboratory findings in monkeys were confirmed by vaccine experience in humans; the number of vaccine-associated paralytic poliomyelitis cases in the population showed a further decline.