Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates the expression of genes associated with adaptation to reduced oxygen pressure. Increased expression of HIF-1alpha gene (HIF1A) has been found in the majority of prostate carcinomas. In addition, the PC-3 prostate cancer cell line has been shown to express the gene even under normoxic conditions. By comparative genomic hybridization (CGH), we have earlier shown that the PC-3 cell line contains a high-level amplification in the chromosomal region harboring the HIF1A gene. Here, we first fine mapped the gene to locus 14q23 by fluorescence in situ hybridization (FISH). The gene was then shown to be highly amplified in the PC-3 cell line. Subsequently, the copy number of the HIF1A gene was studied in 5 other prostate cancer cell lines (LNCaP, DU-145, NCI-H660, Tsu-Pr, JCA-1) and in 117 prostate tumors representing both hormone-dependent and -refractory disease as well as primary and metastatic lesions. No high-level amplifications of the HIF1A gene were found. Additional copies of the gene were seen in all of the cell lines and in 36% of the tumors. There was no association between the tumor type and the copy number alterations of the gene. In conclusion, high-level amplification of the HIF1A gene may explain the overexpression of the gene in the PC-3 prostate cancer cell line. However, such high-level amplification seems to be very rare in prostate cancer.