Differential expression can be used to identify genes that are likely to be involved in the development and progression of cancer. To detect genes whose expression is decreased in prostate cancer, we have combined two methods: suppression subtractive hybridization (SSH) and cDNA library array. Although both methods are potentially powerful, so far they have been inadequately validated. Here we have studied the enrichment of differentially expressed sequences by subtraction and investigated the sensitivity, linearity and reliability of cDNA array hybridization. A subtracted cDNA library was constructed using benign prostatic hyperplasia (BPH) as a tester and a prostate cancer cell line (PC-3) as a driver. Inserts from 386 individual clones were PCR amplified and arrayed on nylon membranes, which were subsequently hybridized with radioactively labelled first-strand cDNA preparations from BPH, PC-3 and several other cancer cell lines. To demonstrate the enrichment of differentially expressed sequences, the number of clones from prostate-specific antigen (PSA) was studied in the subtracted library and in unsubtracted library. Northern analysis was used to confirm the results of cDNA array hybridization. Array hybridization sensitivity and linearity were studied by hybridizing the membranes with cDNA probes containing various amounts of PSA cDNA. The number of PSA was 17-fold higher in subtracted cDNA library compared with unsubtracted cDNA library, demonstrating the enrichment of differentially expressed sequences by the SSH. The detection limit of the array hybridization was found to be 50 pg, corresponding 0.01% of the total poly(A)+ RNA. cDNA array hybridization was linear from 50 pg to 1,000 pg. Of the 386 subtracted cDNA clones analysed, 111 were classified as differentially expressed between BPH and PC-3 by array hybridization. In conclusion, by combining SSH and cDNA array hybridization we were able to detect sequences that are differentially expressed in BPH and PC-3. Further studies will be carried out to determine whether some of them are truly involved in tumorigenesis of prostate cancer.