32P-Postlabeling analysis is a useful assay system for detecting the covalent binding of mutagens and/or carcinogens to DNA. The detection ability of this system has been tremendously enhanced by the incorporation of butanol extraction or nuclease P1 treatment into the experimental protocol. In this study, the sensitivity of adduct detection between these two enhancement methods was compared in vivo or in vitro with 2-aminoanthracene (2AA), 2,4,7-trinitro-9-fluorenone (TNF), and nitrosated coal dust extract (NCDE) using the lung cells of rats. For the in vivo assay, male CD rats were dosed 3 times via intratracheal instillation, whereas for the in vitro study, rat lungs cut into small pieces were treated with test substances for 16 h without exogenous activation. Although, under the conditions tested, both the butanol and the nuclease P1 methods detected DNA adducts caused by all 3 test agents in rat lung cells in vivo or in vitro, a higher adduct detecting ability was found with the butanol enhancement for 2AA and TNF, and with the nuclease P1 enhancement for NCDE. The results suggest that overall the butanol enhancement method is a more sensitive protocol. However, for detecting unknown adduct-forming chemicals, especially when they are present in complex mixtures, both enhancement methods may have to be used.