Epstein-Barr virus infection has been associated with lymphoepithelioma-like carcinoma of the lung in Asian patients. Recently, Epstein-Barr virus proteins or genomic DNAs were detected in pulmonary squamous-cell carcinoma, adenocarcinoma, and undifferentiated small-cell carcinoma in American patients. We studied 23 cases of small-cell carcinoma of the lung for evidence of Epstein-Barr virus infection by in situ hybridization, immunohistochemistry, and polymerase chain reaction methods. Of the 23 cases, 13 cases were primary small-cell carcinoma of the lung and 10 cases were metastatic small-cell carcinoma of the lung to the brain (one case), liver (two cases), and lymph nodes (seven cases). None of the 23 cases was positive for Epstein-Barr virus-encoded small nonpolyadenylated RNA (EBER)-1 by in situ hybridization. By immunohistochemistry, eight cases showed focal positivity for Epstein-Barr virus nuclear antigen-1. The positive immunostaining was focal and was observed in tumor cells, vascular endothelial cells, and lymphocytes, suggesting nonspecific staining. None of the 23 cases was positive for the transactivating immediate-early BZLF1 (ZEBRA) and latent membrane protein (LMP-1). Only one case was positive for the BamHI W region and LMP-1 gene by polymerase chain reaction assay. Some tumor cells in the BamHI W region positive case were also positive for Epstein-Barr virus nuclear antigen-1. Our study indicates that rare cases of American small-cell carcinoma of the lung may contain Epstein-Barr virus-infected cells, but it is unlikely that Epstein-Barr virus plays a role in the tumorigenesis of small-cell carcinoma of the lung.