The use of genetically resistant maize hybrids is the preferred means of control of gray leaf spot, caused by Cercospora zeae-maydis. One problem faced by maize breeders attempting to breed for resistance to gray leaf spot is the high degree of genotype-environment interactions observed in disease trials. In North Carolina gray leaf spot trials conducted at four locations in the western part of the state, we found consistent hybrid-location interactions over the 1995 and 1996 growing seasons. Isolates of C. zeae-maydis from those test locations were evaluated on the same hybrids used in the multilocation testing at a location in central North Carolina that does not have a history of gray leaf spot. The hybrid-isolate interactions observed in the isolate trial mirrored the hybrid-location effects seen in the multilocation testing. Most of the interactions arose from changes in the magnitude of differences between hybrids when inoculated with the isolates rather than from any change in hybrid ranking. Analysis of internal transcribed spacer-restriction fragment length polymorphisms (RFLPs) and mitochondrial rDNA RFLPs of those isolates and others revealed that both type I and type II sibling species of C. zeae-maydis, as well as C. sorghi var. maydis, are isolated from typical gray leaf spot lesions. Breeders should use the most aggressive isolates of C. zeae-maydis to maximize discrimination between genotypes in gray leaf spot trials.