To understand the mechanism in induction of embryogenesis from microspores of Brassica napus, we isolated exhaustively the genes expressed differentially during the early stage of microspore culture. A subtracted cDNA library composed of up-regulated genes during androgenic initiation was produced by suppression subtractive hybridization followed by differential screening by dot blot hybridization, and a total of 136 non-redundant expressed sequence tags were identified. Analysis of the potential functions of the genes showed that 64% of these genes were homologous to known genes, and the remaining ones have not been previously reported to participate in embryogenesis. Many embryo-specific genes were contained in the isolated genes, for example, genes cording lipid transfer protein, napin, cruciferin, oleosin, and phytosulfokine. Real-time RT-PCR analysis for 15 selected genes, which are understood to not be related with embryogenesis, demonstrated that all genes were expressed highly in the early stage of microspore embryogenesis. A few genes also showed higher expression in microspores cultured in non-embryogenic condition or in later stages of embryos. A principal component analysis based on expression profiles of the 15 genes demonstrated that these genes were classified into 2 groups, one characterized by their high expression in initiation of embryogenesis, and the other characterized by their expression in the early to middle stage of embryogenesis. The expressions of these genes were confirmed in zygotic embryos. The identification and characterization of the genes isolated in the present study provide novel information on microspore embryogenesis in Brassica.