Heterogeneity in colony size of the murine pathogen Mycoplasma pulmonis was examined. Subcloning experiments showed that colony size variation resulted from high-frequency genetic changes. About 3% of the colonies from any given subclone were variants, with as much as a fourfold change in colony diameter. When the variants were propagated in liquid broth, their doubling times in logarithmic growth phase reflected the colony sizes obtained on agar. Colony size variation correlated with changes in the electrophoretic properties of the V-1 surface antigen.