Mushroom is the only microorganism cultivated as the crop, and Plerotus ostreatus is one of the most important edible mushrooms. Efficient production of edible mushrooms relies on the precise control of fruiting body development, and an identification of the molecular mechanism of fruiting body development has commercial and scientific importance. In order to identify the developmentally regulated genes during fruiting body development, cDNA libraries were constructed from eight developmental stages of the P. ostreatus. From these libraries, 11,761 expressed sequence tags (ESTs) were generated. Based on these results, we performed macroarray analysis using 1,528 unigene clones at three developmental stages of mycelium, fruiting body and basidiospores. Plasmids isolated from these clones were blotted on the nylon membrane. The isotope-labelled cDNA probes for hybridization of the northern blot were prepared from total RNAs isolated from three developmental stages of mushroom. The 33, 14, 10 unigenes were very highly expressed in mycelia, fruit body and basidiospores, respectively. To confirm expression pattern of these genes, RT-PCR was performed using the total RNA isolated from three developmental stages. Seven genes were successfully amplified in RT-PCR. The expression patterns of the genes were similar with that in macroarray. One of seven genes was identified as a 12kDa heat shock protein and its expression level was very highly at fruiting stage, but not detected at mycelium stage.