The subcellular expression patterns and fluorescence intensities of cytoplasm-targeted, constitutively expressed blue-, cyano-, green-, yellow- and red-fluorescent protein were assessed in a number of transformants of the blast pathogen, Magnaporthe grisea. All transformants grew normally, remained pathogenic on barley, and, except for those expressing blue fluorescent protein, exhibited significant cytoplasmic fluorescence. The exceptionally intense brightness of some strains proved very useful for laser scanning confocal microscope imaging during invasion of host tissues. Acquisition of three-dimensional data sets from intact, individual, pathogen encounter sites in planta were generated during the time course of pathogenesis using non-invasive optical sectioning methods. Confocal and multiphoton microscopy imaging in conjunction with fluorescent protein expression allowed for the real time documentation of fungal colonization within plant cells and tissues with remarkable ease. These methods constitute valuable new tools for the investigation of plant disease.