In this report we present evidence for a novel switch in the ratio of the two major isoforms of the polypyrimidine tract binding protein (PTB) in two related prostate cancer cell lines. The existence of different isoforms of PTB is thought to be the result of alternative splicing. We used UV cross-linking to identify a PTB doublet in the DT3 cell line, which is a rat prostate epithelial cancer line that is androgen-dependent and nonmetastatic. The AT3 cell line, a metastatic, androgen-independent cell line derived from the same tumor as the DT3 cells, was noted here to have a different isoform ratio of PTB. The two most prevalent isoforms of PTB were found to bind to an RNA probe containing a pyrimidine stretch. Western blot analysis demonstrated that these isoforms are indeed expressed differently in the two cell lines and that the observed binding is the result of this differential expression. These two cell lines are derived from the original Dunning prostate tumor, which is a model for studying tumor progression in the prostate. This ratio switch may be an important event in tumor progression in this model system of prostate cancer.