Subtracted cDNA libraries were screened with cDNA macroarrays to isolate larval and pupal stage-specific genes from Aedes aegypti. Of 103 partial cDNAs sequenced from the 4th instar subtracted cDNA library, 62 have counterpart genes in other organisms while 41 of them have no significant similarity to any known genes. Sequences of 116 partial cDNA clones from the pupal subtracted library revealed that 57 belong to unknown genes and 59 have homologous genes in other organisms. Results of cDNA macroarrays showed that 42-50% of randomly selected genes in the subtracted cDNA libraries were differentially expressed. Of the unknown genes, transcripts of 15-19% of the genes were detected in larval or pupal stages, respectively. The results indicate that a subtracted cDNA library in combination with a cDNA macroarray can be used effectively to identify genes expressed in a particular stage.