A change in Y chromosome number is one of the many cytogenetic abnormalities reported in human prostate tumors. However, reports in the literature have varied regarding the frequency of Y loss or gain and the significance of Y aneusomy with respect to the biology of the disease. We have conducted an analysis of the Y chromosome in malignant and benign hyperplastic human prostate epithelium in order to determine whether regional Y loss occurs in prostate cancer. To accomplish this we performed dual-color fluorescence in situ hybridization (FISH) on serial sections of paraffin-embedded prostate tumor tissues using either a Yp (SRY), Ycen (alpha-satellite) or Yq (satellite 3) probe, and an Xcen (alpha-satellite) probe that served as a control for hybridization and nuclear truncation. The results of our FISH analysis demonstrated loss of Yp in the malignant epithelium of 14/40 (35%) prostate tumor sections examined. We also found loss of Yq in 4/40 (10%) of the samples, with one of these exhibiting accompanying Yp loss. The remaining samples, 23/40 (58%), retained both Yp and Yq markers, with no evidence of either Ycen loss or Y gain in any of the tumor samples examined. In addition, Y loss was detected in the benign hyperplastic regions in nearly one-half of the tissue sections that exhibited Y loss in the malignant epithelium. These results demonstrate that regional chromosome Y loss occurs in prostate cancer, that loss of Yp is the most frequent event, and suggest that this loss may in some cases be a precursor to prostate malignancy.