Abstract: To find out if amplification and overexpression of the TRIO gene, encoding a protein with a putative role in cell cycle regulation, is correlated to progression of bladder cancer because previous CGH analysis has shown that 5p amplification is linked to progression of bladder cancer and the TRIO gene maps to 5p15.1- p14. Methods: Fluorescence in situ hybridization (FISH), RNA in situ hybridization and Northern blot analysis were used in this study. Results: FISH analysis of a small tissue microarray showed that the TRIO gene was included in the amplified area in 12 of 14 cases. Furthermore, the FISH study from a tissue microarray containing samples from 1636 bladder tumors suggested that TRIO amplification was closely associated with an invasive phenotype and poor differentiation of the tumor (P<0.0001). TRIO amplification can only be found in 1.5% (7 of 456) of cases with early stage pTaG1/G2 tumors, while it was found in 12.8% (62 of 485) of cases with pT1- 4 bladder cancer. RNA in situ hybridization and Northern blot analysis confirmed that TRIO was overexpressed in tumors. Conclusion: TRIO is frequently amplified and overexpressed in invasive bladder cancer (stage pT1 - 4). The amplification and overexpression of TRIO may have a role in progression of bladder cancer.