Gibberella zeae (anamorph Fusarium graminearum) is the causal agent of Fusarium head blight (FHB) of wheat and barley in the United States. Ascospores forcibly discharged from mature fruiting bodies, the perithecia, serve as the primary inoculum for FHB epidemics. To identify genes important for perithecium development and function, a cDNA microarray that covered 11% of the G. zeae genome was constructed. The microarray was used to measure changes in transcription levels of genes expressed during three successive stages of perithecium development. When compared with vegetative mycelia, 651 (31%) cDNA clones showed changes in transcript levels in at least one of the three developmental stages. During perithecium development, 263 (13%) cDNA clones showed temporal changes in transcript profiles. Transcripts that showed the greatest changes in levels in maturing perithecia belonged to genes in the FunCat main functional categories of cell rescue, metabolism, cell type differentiation, energy, and cellular transport. For genes related to metabolism and cell type differentiation, transcripts showed the highest levels in immature perithecia, whereas for cellular transport-related genes, transcripts showed the highest levels in mature perithecia. This study represents the first large-scale investigation of both spatial and temporal changes in transcript levels during perithecium development. It provides clear evidence that the sexual development in fungi is a complex, multigenic process and identifies genes involved in sexual development of this agriculturally important fungus.