Testicular germ cell tumors (TGCTs) show, for the most part, a characteristic cytogenetic aberration carrying one or more copies of i(12p). Mostly i(12p)-negative TGCTs also show an overrepresentation of 12p11p12 sequences. Critical genes in the amplicon defined by 12p11p12 may be involved in the progression of TGCTs and other tumors. To identify directly the expressed sequence tags (ESTs) involved in the amplicon, we performed comparative genomic hybridization array analysis of five TGCTs demonstrated to exhibit high DNA copy number in this region. The use of arrays spotted with 8,060 IMAGE complementary DNA clones amplified by means of the polymerase chain reaction (PCR) identified 18 amplified ESTs. Most of them were mapped to 12p and the remainder have been mapped to 12. We also identified new testis-associated transcripts by PCR with reverse transcription of the amplified EST sequences. Using combined RNA-array and real-time-beacon PCR analysis, we identified two amplified and overexpressed ESTs. The level of expression in these ESTs was four- to sixfold higher in one tumor sample compared with normal testis. The expression ratios obtained by real-time-beacon PCR analysis correlated with those obtained by RNA-array data analysis, thus validating the techniques used. However, we failed to find an amplified and overexpressed EST commonly overexpressed in all the tumor samples tested, perhaps because the arrays used might only represent approximately 10% of human genes. Therefore we decided to design a 12p array containing 768 ESTs, representing all the 12p-mapped ESTs found in the available databases. Results of comparative genomic hybridization and RNA-array analysis of TGCTs using the 12p array will be presented.