Prostate cancer is the most frequent malignant disease and the second most frequent cause of death due to cancer in men in the Western world. Since serum prostate-specific antigen (PSA) and its subforms show poor specificity in clinical practice, a molecular marker for the detection and discrimination of prostate cancer (PCa) could be of great interest. To investigate the potential significance of genetic aberrations, such as loss of heterozygosity (LOH), in PCa we identified and characterized allelic losses in circulating tumor-associated DNA in blood from patients with localized PCa. Genomic DNA extracted from cell-free plasma of blood samples drawn from 65 PCa patients was analyzed using a panel of 15 polymorphic microsatellite markers mapping to known tumor-suppressor genes. Comparative analyses were performed with a control group of 36 patients with benign prostatic hyperplasia (BPH). In the current study, we demonstrate that PCa patients had higher DNA concentrations in their blood circulation than BPH patients. In the marker panel studied, LOH was more frequently detected in PCa patients (34%) than in BPH patients (22%). The incidence of LOH in the plasma DNA of PCa patients was highest at chromosomal regions 3p24 (THRB, 22%) and 8p21 (D8S360, 22%) in comparison to the BPH control cohort, which frequently showed LOH at loci 8q21, 8p21, 9p21, and 11q22 (D8S286, D8S360, D9S1748, and D11S898, each 6%). These results indicate that microsatellite analysis using plasma DNA may be an interesting tool for molecular screening of PCa patients.