Objective: Since well-differentiated adenocarcinoma cells of the lung (G1 cancer cells) show mild atypia, their differentiation from benign columnar epithelial cells (benign cells) is often difficult based on morphology. We performed discriminant analysis to distinguish benign from malignant cells by measuring 3-dimensional (3D) changes in nuclear luminance. Study Design: Discriminant analysis of 231 atypical cells prepared by bronchial brushing cytology, which were difficult to morphologically classify as benign or malignant, was performed using 100 G1 cancer cells. One hundred benign cells of bronchial brushing cytology specimens served as controls. The number of pixels of the nucleus, the number of focus layers and the level of change in the coefficient of variation (CV) of nuclear luminance between layers (3D-CV) were used as analytic parameters, and benign cells were discriminated from malignant cells based on the Mahalanobis distance. Results: As a result of discriminant analysis using a cutoff value determined in the control group, about 90% of the atypical cells difficult to classify as benign or malignant could be classified. Conclusion: For cells difficult to morphologically classify as benign or malignant, discriminant analysis based on 3D changes in nuclear luminance may be useful. This method can provide objective parameters for cancer diagnosis.