The use of in situ hybridization to show human papillomavirus deoxyribonucleic acid in metastatic cancer cells within lymph nodes. Southern blot hybridization has been used to identify human papillomavirus types in both primary tumors and lymph node metastases. However, this technique requires fresh-frozen tissue and is incapable of localizing deoxyribonucleic acid sequences to specific cell types in the tumor sample. In contrast, in situ hybridization precisely locates viral sequences within tumor cells while preserving cellular architecture. Further, in situ hybridization requires only small samples of formalin-fixed, paraffin-embedded tissues. Five lymph nodes (from four patients) containing metastatic cervical squamous tumor cells (identified with hematoxylin and eosinophil staining) were analyzed with in situ hybridization techniques with human papillomavirus type 16 deoxyribonucleic acid probes labeled with sulfur 35. The primary cervical cancer from all four patients had been shown to contain human papillomavirus type 16 sequences by Southern blot. Three specimens from two patients clearly showed the presence of human papillomavirus type 16 sequences within the nuclei of metastatic tumor cells, whereas two specimens were nondiagnostic most likely as a result of the small volume of cancer relative to the size of the lymph node. This information indicates that it is the tumor cells themselves that contain viral deoxyribonucleic acid and provides additional evidence linking human papillomavirus with cervical carcinogenesis. 