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To identify genes associated with morphological phenotypes of human bladder transitional cell carcinoma, we used suppression subtractive hybridization (SSH) to create a subtractive cDNA library of two established cell lines, BLZ-211 and BLS-211, derived from a patient with transitional cell carcinoma of the bladder, then to screen for differentially expressed genes. Real-time reverse transcription-polymerase chain reaction was used to further confirm the selected differentially expressed genes. Forward and reverse subtractive cDNA libraries yielded 168 and 305 putative clones, and among them more than 90% contained the inserts.After differential screening, 36 different transcripts were obtained from 64 cDNA clones of a forward and reverse subtraction library. Among them, 17 were identified as known genes by homology, for example,Vimentin, Keratin7, DDH and UCH-L1. The remaining 19 were unknown expressed genes, and were collected as new expressed sequence tags by the GenBank dbEST database with the accession numbers DR008207,DR010178, DR159652-DR159660, DY230447-DY230448, and DY505708-DY505713. Their function will be studied further. Thus, SSH appears to be a useful technique for identifying differentially expressed genes between cell lines or clones. Our results, as revealed by SSH, also suggest that differences in gene expression of cytoskeletal proteins might contribute to the different morphologies in BLZ-211 and BLS-211 cells.