It has been suggested that ingestion of polycyclic aromatic hydrocarbons (PAHs) may contribute to the high incidence and mortality of esophageal cancer in Linxian, China. To explore this relationship a semiquantitative immunohistochemical staining method was developed for localization of PAH-DNA adducts. Nuclear color intensity (bright field average pink intensity per nucleus for >1000 cells) was measured using the ChromaVision Automated Cellular Imaging System (ACIS). Paraffin-embedded sections of cultured human keratinocytes exposed to increasing concentrations of 7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) were incubated with BPDE-DNA antiserum and served as an internal positive control (standard curve). Values for nuclear staining intensity correlated directly with BPDE exposure concentration (r(2) = 0.99) and were reproducible. DNA adduct levels determined by BPDE-DNA chemiluminescence immunoassay in DNA from BPDE-exposed keratinocytes, correlated with BPDE exposure concentrations (r(2) = 0.99), showing that nuclear staining intensity determined by ACIS correlated directly with BPDE-DNA adduct levels determined by chemiluminescence immunoassay. The ACIS methodology was applied to 5 human samples from Linxian, and significantly positive nuclear PAH-DNA adduct staining was observed in this group when compared with esophageal tissue from 4 laboratory-housed monkey controls and 6 samples obtained at autopsy from smokers and nonsmokers in the United States. Nuclear PAH-DNA staining was absent from Linxian samples when serial sections were incubated with normal rabbit serum (negative control) and was significantly reduced on incubation with BPDE-DNA antiserum absorbed previously with the immunogen BPDE-DNA. These results appear to support the hypothesis that high PAH exposure levels may be etiologically associated with the development of esophageal cancer in Linxian.