Microsporidia form specialized environmentally resistant spores that infect almost all vertebrate and invertebrate species. They are a unique member of the amitochondrial protozoa, with a phylogenetic kinship to fungi. Although the mode of transmission to humans remains unproven, it is suspected that human microsporidia are environmentally transmitted via ingestion or direct inoculation of spores, which are shed in large numbers into water and the environment from infected hosts. This study was conducted to assess the growth of human microsporidia spores and optimize in vitro spore growth in cell culture. The optimized cell culture systems were used to design an in vitro viability assay, so that work could begin to determine spore viability after exposure to chlorine or other disinfectants.