A vital staining assay using Hoechst 33342 and propidium iodide was developed for the determination of viability of Enteromyxum leei (Myxozoa), a causative agent of myxosporean emaciation disease. Using this in vitro viability assay combined with in vivo infectivity test, the survival of developmental stages of E. leei in seawater was evaluated. Developmental stages of E. leei were freshly isolated from infected tiger puffer Takifugu rubripes and incubated in filtered natural seawater at 20ºC for 0, 6, 12 and 24 h. The in vitro staining assay showed a time-dependent decrease in viability, and no viable stages were detected at 24 h. In the first trial of in vivo infectivity, naïve tiger puffer were exposed to E. leei suspensions which were incubated in seawater for 0-48 h. In the second trial, naïve grass puffer T. niphobles were fed E. leei incubated for 0-24 h. After rearing for 3-4 weeks, infection with E. leei was detected in fish challenged with the parasite incubated for 24 and 6 h in the first and second trials, respectively. Results of the in vitro and in vivo assays showed that the survival time of developmental stages of E. leei in seawater was variable but estimated less than 24 h.