We are investigating development of Fusarium graminearum on and in barley florets and other organs using a transformed strain of the fungus containing a constitutively expressed gene for green fluorescent protein (GFP). The GFP strain fluoresces green when viewed by fluorescence microscopy under blue or blue-violet incident light. The GFP strain is easier to see in inoculated living florets, leaf or coleoptile tissues (as viewed by epifluoresence microscopy) than are wild strains (viewed by conventional light microscopy). To remove surface mycelium which can obscure fungus development within underlying tissues, a 2 to 1 (v/v) solution of cellulose acetate in acetone is applied to the tissue surface, allowed to dry for a few minutes, then stripped away, leaving the tissue surface intact and free of mycelium. In initial experiments, inoculum in the form of mycelium growing on mung bean agar blocks (2x12x0.5 mm) was applied to cut ends of detached leaves, coleoptiles, the palea, and the lemma from Robust (susceptible) and Chevron (partially resistant) barley. After 24 hr, the block was removed. The fungus grew both into and on top of the inoculated tissues. By three days after inoculation, hyphae within tissues grew 2.9-3.1 mm in leaves, 1.2 - 1.5 mm in coleoptiles and paleae, but less than 0.2 mm in lemmae (as measured from the cut ends to the advancing hyphal front). In leaves, the distance was increased to 4.5 - 5.3 mm if the inoculum block was left in place 48 hr instead of 24 hr. Hyphae within tissues were subcuticular and intercellular. Plant cells in colonized leaves remained green 2-2.5 mm behind the advancing hyphae front. Tissues more than 2.5 mm from the front became chlorotic, losing the red fluorescence of chlorophyll. On the surface of the tissue, mycelium advanced about 0.3 mm ahead of the underlying subcuticular and intercellular hyphae. Hyphae appeared to grow into and out of leaf stomates. The results show that fungus development was limited in paleae, lemmae and coleoptiles compared to development in leaves, that exogenous nutrients can increase amount of colonization in leaves, and that development was the same in tissues of Robust and Chevron. Based on experience gained in these experiments, we will use the GFP strain to monitor pathways of infection from spot inoculations on exposed surfaces of the palea and lemma of intact barley heads, and to investigate the probable role of anthers in promoting head colonization.