Expressed sequence tag (EST) analysis of tobacco (Nicotiana tabacum) anthers was performed for 200 cDNA clones randomly chosen from a cDNA library representing transcripts of developing tobacco anthers. Only 21 clones showed significant homology to the nucleotide sequences in databases. Most of these belonged to the housekeeping genes, such as for ribosomal protein L7, histone H3.2, histone H2B-2, and amino acid transporter. For the ESTs showing high levels of similarity to previously reported anther-specific genes, RNA blot hybridization was performed to characterize expression patterns of the gene in the anther, Clones 4C3 and 6Cf showed anther-specific expression. 4C3expressed strongly in the early stage, and 6C1 mainly expressed during the late stage of anther development.