L20B cells are genetically engineered for the cell surface expression of the poliovirus receptor CD155 and are commonly used for the isolation of poliovirus as well as other enteroviruses. The current report documents a thin sectioning electron microscope analysis of uninfected and poliovirus infected L20B cells and indicates L20B cells are chronically infected with retrovirus-like particles. The retrovirus-like particles were round or ovoid in shape and had two forms: a cytoplasmic form and a budding form. The cytoplasmic form had a mean diameter of 52.0 nm with a mean core diameter of 23.9 nm. The budding forms showed evidence of further maturation as they had an additional outer layer and a larger core; these forms had a mean diameter of 75.1 nm and a mean core diameter of 46.7 nm. The findings indicate care is needed in the use of L20B cells for certain virus isolation studies and emphasize the importance of electron microscope studies as an adjunct to the development of diagnostic virology protocols.