Three different cultures of BCG propagated for over 2 years in a liquid medium containing Tween 80 and albumin were found to differ in several of their intrinsic properties. Cultures of the three strains were found to consist of morphologically heterogeneous populations-each culture being made up of three main colonial types-spreading, intermediate, and non-spreading. The percentage distribution of colonial types was characteristic for each culture and remained constant during cultivation in liquid media. Injection of the various cultures into mice and guinea pigs resulted in a self-limited disease. The distribution, extent, and duration of the lesions were also characteristic for each culture. Both the spreading and non-spreading substrains derived from the various cultures exhibited the degree of attenuation of virulence characteristic of the parent strain.