This is the first report using DNA molecular probe technology to distinguish between recurrent tumor and a second primary malignancy in a patient. Tumor DNA was extracted from squamous cell carcinoma of the cervix at the time of radical hysterectomy. Eighteen months later a squamous cell cancer was found in a vaginal apex biopsy from which DNA was extracted. Tumor DNA from both lesions was subjected to restriction enzyme digestion and DNA molecular hybridization with human papillomavirus (HPV) probes. Although both lesions were positive for HPV 16, their respective restriction enzyme patterns had different HPV genetic arrangements, thereby demonstrating their distinctness.