A total of 65 isolates of Vibrio cholerae, serotypes other than O--1, have been recovered from water, sediment, and shellfish samples from the Chesapeake Bay. Isolations were not random, but followed a distinct pattern in which salinity appeared to be a controlling factor in V. cholerae distribution. Water salinity at stations yielding V. cholerae (13 out of 21 stations) was 4 to 17 0/00, whereas the salinity of water at stations from which V. cholerae organisms were not isolated was less than 4 or greater than 17 0/00. From results of statistical analyses, no correlation between incidence of fecal coliforms and V. cholerae could be detected, whereas incidence of Salmonella species, measured concurrently, was clearly correlated with fecal coliforms, with Salmonella isolated only in areas of high fecal coliform levels. A seasonal cycle could not be determined since strains of V. cholerae were detectable at low levels (ca. 1 to 10 cells/liter) throughout the year. Although none of the Chesapeake Bay isolates was agglutinable in V. cholerae O group 1 antiserum, the majority for Y-1 adrenal cells. Furthermore, rabbit ileal loop and mouse lethality tests were also positive for the Chesapeake Bay isolates, with average fluid accumulation in positive ileal loops ranging from 0.21 to 2.11 ml/cm. Serotypes of the strains of V. cholerae recovered from Chesapeake Bay were those of wide geographic distribution. It is concluded from the data assembled to date, that V. cholerae is an autochthonous estuarine bacterial species resident in Chesapeake Bay.