A cDNA library containing 2409 singletons was constructed from whole silkworm pupae (Bombyx mori) In addition, the types of genes overexpressed in pupa were analyzed. These genes contained 79 types of proteins with the exception of enzyme, mitochondrial DNA, andribosomal protein. Also analyzed were the expression and nonexpression of open reading frame (ORF) sequences in Escherichia coli. cDNA sequences were compared to the silkworm (B. mori) genome in the GenBank database and the silkworm cDNA database including the SilkBase and KAIKOBLAST databases and 498 novel expressed sequence tags (ESTs) and 217 unknown ESTs were found. After comparison with all available ORF-complete mRNA sequences from the same organism (fruitfly, mosquito, and apis) in the RefSeq collection, 1659 full-length cDNA were identified. In addition, the structure of silkworm mRNA was analyzed, and it was found that 66.8% of silkworm mRNA tailed with poly(A) contained the highly conserved AAUAAA signal and the signal located 10-17 nucleotides upstream of the putative poly(A). Finally, the composition of nucleotides in promoter region for all ESTs was surveyed. The results imply that the TTTTA box may possess some functions in regulating transcription and expression of some genes.