The clinical course of patients with thymoma varies widely despite its histologically benign appearance. Treatment decisions are based on local invasion and the extent of resection. Because some patients have more aggressive tumors, the prognostic significance of flow cytometric (FCM) analysis of nuclear DNA content was examined. Adequate tissue from paraffin-embedded blocks was available for 25 patients. Using FCM, the percentage of cells in S-phase (%S) and the ploidy, based on the DNA index (DI), were determined. The mean patient age was 52 years, with a female-to-male ratio of 1.3:1 and a median follow-up of 64 months. Seventeen patients underwent total tumor resections, and 12 also received radiation therapy. Eight patients underwent subtotal resections, with five receiving radiation therapy (with or without chemotherapy) and three receiving chemotherapy alone. Based on invasion and intrathoracic dissemination, the tumors were classified into four stages. The mean %S was 5.6. There was no relationship observed between %S and patient outcome. The 5-year disease-free survival rate was 85% for the 16 patients with diploid (DI = 1) tumors and 33% for the 9 patients with aneuploid (DI more than 1) tumors (P less than 0.002). Similar significant differences were observed by stage and extent of surgery. For those who had total resection (n = 17), the disease-free survival rate was 89% when DI equaled 1 and 50% when DI was more than 1 (P = 0.01). Although the numbers studied were small, when stage, histologic findings, and type of surgery were subdivided by DI, a higher incidence of relapse was associated consistently with aneuploidy. The DI appears to be a useful prognostic parameter for identifying patients at high risk of relapse.