Gray leaf spot (GLS) disease of maize (Zea mays L.), caused by the fungus Cercospora zeae-maydis, causes significant corn yield losses in Virginia and other mid-Atlantic states. A new greenhouse assay method with filter paper discs of C. zeaemaydis mycelia has been developed to evaluate corn germplasm for resistance to GLS. Mycelial inoculum obtained from cultures of mycelia in liquid malt media was pipetted at 100 ul samples onto each filter paper disc which was then adhered to the lower leaf surface by transparent tape. The inoculated corn seedlings were placed in a moist plastic chamber with high relative humidity provided by a humidifier. The first macroscopic symptoms induced by this inoculation method appeared 3 days after inoculation. This new inoculation method with mycelial discs was used on five corn genotypes (VA14, B68, PA875 , B73, and M017) to screen resistance to GLS disease. With this inoculation method, resistant and susceptible inbreds were easily differentiated based on lesion type. Resistant inbreds including VA14, B68, and P A875 were characterized by water-soaked appearance or small chlorotic flecks while susceptible inbreds like B73 and M017 were characterized by more extensive necrosis. Necrotic area under the mycelial disc was a good indicator for disease severity. However, the percent leaf area under discs affected by mycelia which reflected the total host responses was not appropriate to indicate disease severity. The effects of plant physiological factors on the expression of resistance to GLS was also investigated. Placing mycelial discs on lower leaf surfaces induced more responses than placing on upper leaf surfaces. Inoculation of lower older leaves induced more severe lesions than inoculation of upper leaves. The effect of cercosporin was investigated by inoculating corn seedlings with cercosporin-producing mycelia and with non-cercosporin containing mycelia. The former induced much more severe host response than the latter. Conidiation of C. zeae-maydis was examined with the mycelial inoculation method in the greenhouse. Conidiophores were found emerging from stomata as early as 15 days after inoculation in B73 and M017 and limited only to necrotic tissue. No conidiation was observed in resistant genotypes VA14, B68 and PA875.