The oyster mushroom (Pleurotus ostreatus) is one of the most important edible mushrooms worldwide. The mechanism of P. ostreatus fruiting body development has been of interest both for the basic understanding of the phenotypic change of the mycelium-fruiting body and to improve breeding of the mushrooms. Based on our previous publication of P. ostreatus expressed sequence tag database, 1,528 unigene clones were used in macroarray analysis of mycelium, fruiting body and basidiospore developmental stages of P. ostreatus. Gene expression profile databases generated by evaluating expression levels showed that 33, 10, and 94 genes were abundantly expressed in mycelium, fruiting body and basidiospore developmental stages, respectively. Among them, the genes specifically expressed in the fruiting body stage were further analyzed by reverse transcription-polymerase chain reaction and Northern blot to investigate temporal and spatial expression patterns. These results provide useful information for future studies of edible mushroom development.