We previously proposed a new assay using the cytokinesis-block micronucleus (MN) technique to estimate the fraction of cells undergoing mitosis in vitro [dividing fraction (DF)], potential doubling time (Tpot), and radiosensitivity (in terms of MN frequency) of human tumors. In the present study, we applied this technique to primary lung cancers to evaluate their biological characteristics, and the assay results for the proliferative activity were compared with the treatment outcome. Tumor tissues were disaggregated to single cells, which were cultured in the presence of cytochalasin B after (or without) radiation. At intervals, the proportion of multinucleate cells (its maximum value is the DF), the average number of nuclei/cell, and MNs in binucleate cells were scored. The Tpot was the extrapolated time for the nuclei:cell ratio to reach 2.0. Of the 71 tumor samples obtained, the DF and Tpot were evaluable in 61 (86%), and the MN frequency was evaluable in 52 (73%). The median DF and Tpot values were 23% and 7.7 days, respectively, for adenocarcinoma (n = 41), 26% and 8.9 days for squamous cell carcinoma (n = 13), 27% and 6.5 days for large cell carcinoma (n = 3), and 30% and 7.0 days for small cell carcinoma (n = 4). There was no significant difference in the mean DF or Tpot values according to the histological type or disease stage. The mean MN frequency after 2 Gy of radiation (minus the 0 Gy frequency) was 0.15 for adenocarcinoma, 0.17 for squamous cell carcinoma, 0.16 for large cell carcinoma, and 0.20 for small cell carcinoma. The MN frequency after radiation was positively correlated with both the DF and the baseline (at 0 Gy) MN frequency. In non-small cell lung cancer, a DF above the median was associated with an increased recurrence rate after operation, and the Tpot was correlated with the time until relapse in patients who developed recurrence. Although the clinical significance of the MN frequency needs to be clarified in future studies, the DF and Tpot determined by this assay appear to be good parameters of tumor proliferative activity.