DNA/RNA flow cytometry studies were performed on the spinal fluid samples of thirty patients with acute leukemia or lymphoma at the time of clinical central nervous system relapse, and compared with similar studies of 56 patients (98 specimens) who had leukemia in remission with no evidence of CNS disease. Twelve of the 30 patients with CNS involvement had cells with abnormal DNA content in the spinal fluid (40%); the remaining eighteen had cells with diploid DNA content. In the group of 18 with diploid DNA, 10 had other abnormalities detected by flow cytometry. These included eight patients with acute leukemia who had cells with high RNA content, and two patients with non-Hodgkin's lymphoma who had markedly increased proliferation. Of the 22 patients studied by conventional cytology nine were negative for malignant cells, and in eight of these patients flow cytometry studies of DNA/RNA demonstrated abnormalities. Common ALL antigen was demonstrated by flow cytometry in three out of five cases studied. Thus, abnormal DNA content, increased RNA content, increased proliferation and/or expression of the cell surface antigen CALLA identified CNS relapse by flow cytometry in 22 of 30 patients with acute leukemia or lymphoma. The technique appears to be at least as sensitive as conventional cytology and identifies CNS relapse in some patients with negative cytology.