Traditional polymerase chain reaction (PCR) amplification of multiple microsatellite markers to detect microsatellite instability (MI) is labor intensive and may be marker dependent. An arbitrarily primed polymerase chain reaction (AP-PCR) is a modification of PCR that generates a genomic DNA fingerprint using a single, arbitrarily chosen primer which is useful in the detection of somatic genetic alterations. We hypothesize that AP-PCR of an AluI DNA restriction digest, which we term Alu/AP-PCR, identifies genomic instability as well. In order to test this hypothesis, we correlated Alu/AP-PCR fingerprints with analyses of traditional PCR amplified microsatellite markers using paired germline and tumor DNA samples obtained from 68 patients with ovarian cancer. The microsatellite markers tested included dinucleotide, trinucleotide, and tetranucleotide repeats. We found that MI is more common in ovarian cancer than previously thought. We estimate a minimum incidence of MI at 37% based on the 10 traditional markers we tested, to an incidence of 53% based upon our Alu/AP-PCR analysis. All cases of MI were associated with an abnormal Alu/AP-PCR banding pattern. Both MI, detected by polymorphic markers (P=0.03), and an abnormal Alu/AP-PCR pattern (P=0.01) were significantly associated with the occurrence of a second primary malignancy in the same patient. In addition, abnormal Alu/AP-PCR patterns were associated with higher grade lesions (P=0.02), and family history of cancer (P=0.009). These findings suggest: (1) MI may play an important role in ovarian carcinogenesis, and (2) Alu/AP-PCR is a novel technique for identification of genomic instability.