A collection of mycoplasma-like organisms (MLOs) was maintained in plant tissues micropropagated in vitro. MLO-infected plants included Chrysanthemum frutescens L. with chyrsanthemum yellows disease, Gladiolus sp. L. with “germ fins,” Hydrangea macrophilla (Thunb.) DC. with virescence, Rubus fruticosus L. with rubus stunt, and periwinkle [Catharanthus roseus (L.) G. Don] singly infected by the following MLOs: Italian periwinkle virescence, chrysanthemum yellows, North American aster yellows, Italian periwinkle stunt, American periwinkle little leaf. Shoots micropropagated in vitro exhibited symptoms of little-leaf and/or abnormal proliferation of axillary shoots resulting in “witches' broom” appearance that resembled symptoms in grafttransmitted greenhouse-grown or naturally infected field-collected plants. These symptoms, typical of infection by MLOs, were not observed in micropropagated healthy shoots of the same plant species, and, compared with the healthy ones, varied with MLO strain and host plant species. Dot hybridizations with a nonradioactive cloned DNA probe provided evidence for the presence of MLOs in propagated tissues through serial subcultures.