p53 mutations are common genetic alterations in lung cancers and usually result in p53 protein accumulation in tumor cells. Sputum is noninvasive to collect and ideal for screening p53 abnormalities. This study was to determine the feasibility of detecting p53 protein accumulation in sputum cells using an immunofluorescence assay. Sputum samples were collected from 58 Chinese subjects, including 16 lung cancer cases from Xuan Wei (XW) County exposed to coal smoke, 25 mostly tobacco smoke-related cases from Zhengzhou City, and 17 XW controls. The p53 protein accumulation in tumor cells and some atypical cells was detected in 56% (9/16) of the coal smoke-exposed XW cases, 44% (11/25) of the Zhengzhou cases and none (0/17) in controls. The p53+ tumor cells were confirmed morphologically by Papanicolaou staining after the p53 immunofluorescence assay. This is the first report of successfully using an immunofluorescence assay to detect p53 protein accumulation in sputum.