Amplification of 6 p22.3 is one of the most frequent chromosomal alterations in high grade and invasive urinary bladder cancer. In order to determine amplification levels of all known genes inside the 1.6 kb core amplicon, we constructed a small tissue microarray (TMA) from 9 primary bladder cancers and 4 bladder cancer cell lines with known 6p22 amplification, and analyzed it with a panel of 16 overlapping FISH probes constructed from bacterial artificial chromosomes (BACs). The highest amplification rates were observed for the transcription factor E2F3 and the adjacent gene NM_017774, the function of which is not known. For a more detailed analysis of these genes, additional large section analysis was done in 19 primary bladder cancers and 18 bladder cancer cell lines. It showed that E2F3 and NM 017774 were always coamplified, but amplification levels in terms of the number of gene copies were slightly higher (16-19 copies per nucleus) for E2F3 as compared to NM_017774 (13-15 gene copies). Our study demonstrates that E2F3 and NM_017774 are located on the top of the 6p22.3 amplicon in bladder cancer. It remains to be studied which one of the two genes drives 6p22 amplification, or if both genes contribute jointly to the aggressive features of 6p22 amplified bladder cancers.