The prognostic significance of DNA index (DI), S-phase fraction, and heterogeneity determined by flow cytometric DNA analysis was assessed in a prospective study of 249 newly diagnosed transitional cell carcinomas of the bladder. The median observation time was 4.8 years. A total of 456 subpopulations were detected. The S-phases could be estimated in 299 subpopulations. A DI > 1.25 or an S-phase above 9.7% were strongly correlated to invasiveness. One hundred and ten patients were treated with transurethral resection (TUR). Relapse-free survival could not be predicted by the DNA-derived parameters. Univariate analysis of survival showed prognostic significance of diploidy (0.98 < DI < or = 1.02, P = 0.02), hypotetraploidy (1.50 < DI < or = 1.96, P = 0.002), and S-phase size (P = 0.008). Multivariate analysis pointed to the T-classification (RR = 1.64) and hypotetraploidy (RR = 1.57) as prognostic parameters for survival of TUR-treated patients. One hundred and thirty-nine patients received radiotherapy (RT). A significantly better response was found for tumors with a subpopulation with a hypertetraploid DNA content (DI > 2.04, P = 0.05), and a significantly worse response for subpopulations with a maximum S-phase > 24.5% (P = 0.04). T-classification and histological grade had no predictive value. A logistic regression analysis indicated an estimated probability of response to RT of 77% for tumors with a DI > 2.04 and an S-phase < 24.5%, whereas tumors with a DI < 2.04 and an S-phase > 24.5% had only a 28% probability of response. The poor response to RT, predicted by an S-phase > 24.5%, translated into a poor survival, whereas the better treatment response found for patients with a DI > 2.04 did not result in a longer survival. Multivariate analysis pointed to S-phase (RR = 1.70), T-classification (RR = 1.60), and grade (RR = 0.65) as independent prognostic parameters for survival of RT-treated patients.