Investigations regarding the prognostic value of DNA content (ploidy) and proliferative characteristics [percentage of cells in S-phase or S-phase fraction (SPF)] have been greatly facilitated by the application of flow cytometry (FCM) using nuclei isolated from paraffin-embedded tissue. We have applied this technique to tumor sections from patients presenting with non-small-cell lung cancer (NSCLC) in 1980 and 1981. From 67 out of 115 patients material of sufficient quantity and quality was obtained to perform DNA-FCM. A multivariate analysis including stage of disease (UICC), age, tumor histology and treatment modality was performed to examine the prognostic significance of DNA-FCM in NSCLC. Aneuploidy was found in 65% of cases. In our study, the DNA content was not related to histology, stage of disease or treatment modality, nor to the length of survival (log rank test P = 0.62). Calculation of SPF was possible in 49/67 cases. The SPF was not related to histology, stage of disease or treatment modality, but a significant prognostic value was found for survival; patients with a high SPF died earlier (P = 0.04) and this was especially true for squamous cell carcinoma (P = 0.02). This study demonstrates the prognostic importance of DNA-FCM-derived information in NSCLC using a multivariate analysis; however further prospective studies in larger patient populations are needed.