Lepidopteran larvae were sampled in the field to determine levels of infection by the gypsy moth, Lymantria dispar (L.), fungal pathogen, Entomophaga maimaiga Humber, Shimazu & Soper. Lepidopteran larvae were reared from 7 plots in Virginia in which moderate density gypsy moth populations simultaneously exhibited from 40.8 to 97.5% E. maimaiga infection. From a total of 1,511 larvae from 52 species belonging to 7 lepidopteran families in 4 superfamilies, only 2 individuals, 1 of 318 forest tent caterpillars, Malacosoma disstria Hiibner (0.3% infection), and 1 of 96 Catocala ilia (Cramer) (1.0% infection), became infected by entomophthoralean pathogens. Results from genomic DNA probes and bioassays confirmed that E. maimaiga had caused these infections. Laboratory studies yielded infection over a greater diversity of species, and percentages of infection from laboratory studies were higher than findings from the field for the 1 species infected in both the laboratory and field. At all sites, the gypsy moth nuclear polyhedrosis virus also was found, occasionally causing epizootics, but viral occlusion bodies were never found in nontarget Lepidoptera that died. In addition, 279 nontarget Lepidoptera belonging to 34 species in 8 families were collected and reared from areas with low density native gypsy moth populations, and E. maimaiga infections were not found in these nontarget hosts, although E. maimaiga was active in gypsy moth populations. A survey of lepidopteran cadavers collected from 1989 to 1995 containing entomophthoralean spores documented E. maimaiga infections in 3 species of lymantriids. The Lepidoptera-specific North American endemic entomopathogen Entomophaga aulicae (Reichardt in Bail) Humber, which is morphologically identical to E. maimaiga, was found in 1 geometrid species, 1 notodontid, 2 species of arctiids, and 1 introduced lymantriid, but in none of the gypsy moth larvae tested. Our results demonstrate that data from laboratory bioassays are poor estimates for predicting nontarget impact.