Lyophilized cultures of V. cholerae 569B slowly lose their virulence for neonatal and adult mice during long term storage. Following a single passage in orally infected 6-day old mice, a highly virulent strain (designated 569B/MP) was isolated. This organism causes rapidly fatal intestinal infections in 6-day old mice; large numbers can be isolated in pure culture from the intestinal fluid. Freezing and storage at -60 degrees of dead animal provides a simple means of maintaining the high virulence of the culture over a period of at least 9 months. This strain produces choleracic symptoms and death in approx. 50% of adult mice following oral infection. Large numbers of viable organisms may also be isolated from the small intestine over a period of at least 40 h. These criteria have been used as a basis for assessing protection against cholera infection induced by immunization with living or heat-killed V. cholerae given orally or intraperitoneally.