This report describes flow cytometric identification, cell and molecular characterization of two E. intestinalis subpopulations isolated from cell culture propagated, Percoll-purified spore suspensions. The results of the molecular analysis of both spore subpopulations indicated that both populations possessed E. intestinalis 16S rRNA and ¿-tubulin genes. However, only one subpopulation was able to infect RK-13 cells. Researchers should be aware that there is inherent variability in the viability of microsporidia spores propagated using cell culture. Includes 11 references.