Cotton bolls were inoculated with a green fluorescent protein (GFP)-expressing Aspergillus flavus (strain 70) to monitor fungal growth, mode of entry, colonization of cottonseeds and production of aflatoxins.  The GFP strain and the wild type did not differ significantly in pathogen aggressiveness as indicated by similar reductions in inoculated locule weight.  GFP fluorescence was at least 10 times higher than the blue green yellow fluorescence (BGYF) produced in response to infection by A. flavus.  The GFP produced by the strain made it possible to identify and monitor specific plant tissues colonized by the fungus.  For example, the inner seed coat and cotyledon were colonized by the fungus within 72 hours of inoculation and the mode of entry was invariably through the porous chalazal cap in intact seeds.  The amount of GFP fluorescence was shown to be an indicator of fungal growth, colonization and, to some extent, aflatoxin production.  The A. flavus strain expressing GFP should be very useful for rapidly identifying cotton lines with enhanced resistance to A. flavus colonization developed through genetic engineering or traditional plant breeding.  In addition, development of GFP expressing A. flavus strain provides an easy and rapid assay procedure for studying the ecology, etiology, and epidemiology of cotton boll rot caused by A. flavus resulting in aflatoxin contamination.