ABSTRACT: A nested polymerase chain reaction (PCR) was developed for detection of the microsporidian parasite Enterocytozoon salmonis in biological samples (blood buffy-coat cells, feces, tissues, lymphocyte cultures) of chinook salmon Oncorhynchus tshawytscha. A major second-round PCR product of 407 bp was readily identifiable in ethidium bromide-stained agarose minigels. An internal probe was used to verify the identity of the amplified product by non-radioactive (digoxigenin-based) Southern blotting; final confirmation was made by DNA sequence analysis. A dilution study using infected lymphocytes from in vitro cultures indicated that a single round of PCR (35 cycles) was able to detect E. salmonis DNA from approximately 1000 infected cells. Sensitivity was increased with the full nested PCR (35 additional cycles), which detected parasite DNA from <=10 infected lymphocytes. The specificity of the PCR was assessed with a panel of microsporidian and myxosporean DNAs. In an experimental infection study, E. salmonis DNA was detected in blood, feces, and tissues of infected chinook salmon but not in uninfected control fish.