Spores of two microsporidia, Nosema pyrausta (from the European corn borer, Ostrinia nubilalis) and N. furnacalis (from the Asian corn borer, O. furnacalis) were harvested from laboratory-reared O. nubilalis caterpillars and purified by centrifugation through Percoll. Conditions permitting in vitro germination were defined for both species and found to be different. N. pyrausta spores were incubated in 0.1 N KOH for 30 min, recovered by centrifugation, and resuspended in 1 ml of an equal mixture of 1% low melting point (LMP) agarose and L-15B medium at 37 degrees C to induce germination. N. furnacalis spores were first washed in 10 mM Na2EDTA in 1 mM Tris base, pH 7.5, exposed to 0.01 N KOH in 0.17 M KCl for 30 min, centrifuged, and germinated in 1 ml of an equal mixture of 1% LMP agarose and 0.17 M KCl in 10 mM Na2EDTA (pH 8), at 37 degrees C. Eighty to 90% of the spores of each species germinated. Germinated spores were pipetted into a casting mold. Before electrophoresis, agarose blocks were incubated 48 hr at 50 degrees C in 10 mM Tris base/100 mM Na2EDTA, pH 7.8, with 1 mg/ml proteinase K and 1% N-laurylsarcosine to release the chromosomal DNA from sporoplasms. After pulsed-field electrophoresis, ethidium bromide staining revealed 13 chromosomal bands ranging in size from 1390- to 440-kb pairs and 1360- to 440-kb pairs in N. pyrausta and N. furnacalis, respectively. The difference in size estimates of corresponding chromosomes in the two species was not more than 60-kb pairs.