Existing evidence supports the hypothesis that human papillomavirus (HPV) may play an etiologic role in the malignant transformation of squamous epithelial cells. Although HPV DNA has been identified in a high proportion of squamous cell carcinomas (SCC) of the cervix, anorectum, skin, and upper airways, few studies have tested for HPV in SCC of the lung. To confirm the presence of HPV in lung SCC, we tested for HPV DNA extracted from formalin-fixed tissues of 34 patients by polymerase chain reaction (PCR). DNA amplification was performed using HPV L1 consensus sequence primers (MY11 and MY09; Perkin-Elmer Cetus, Norwalk, CT) which recognize a broad spectrum of HPV types including 6, 11, 16, 18, 31, and 33, among many other known types, as well as at least 20 other unidentified types. PCR products were analyzed by agarose gel electrophoresis and Southern blot hybridization with [32P]-labeled generic HPV probes. HPV DNA positive cases were subsequently analyzed by slot-blot hybridization of the PCR products with specific probes for HPV types 6, 11, 16, 18, and 33. HPV type 18 was detected in two cases, including one case from a 44-year-old female and one from a 64-year-old male, with the remaining 32 cases negative. In situ hybridization for HPV DNA failed to detect HPV types 6/11, 16/18, or 31/33/35 in any of the cases. We conclude that a small proportion of cases of primary pulmonary SCC test positive for HPV type 18 but that the great majority of cases are not associated with HPV.