In the majority of patients with acute myeloid leukemia (AML) immature leukemic subpopulations expressing myeloid markers and terminal deoxynucleotidyl transferase (TdT) are present. The normal counterparts of these double-positive cells are rare in bone marrow (BM) (< 0.03%; if they occur at all) and are not detectable in peripheral blood (PB). In 14 patients with TdT+ AML at diagnosis, we have performed a prospective follow-up study to monitor the myeloid-marker+, TdT+ cells during and after chemotherapy. One patient did not obtain complete remission (CR), a second patient relapsed under therapy, whereas the other 12 patients were in cytomorphological CR at the end of chemotherapy. During subsequent follow-up, seven of these 12 patients developed one or two relapses (total of ten relapses). Nine of these ten relapses were preceded by a gradual increase of myeloid-marker+, TdT+ cells in BM and PB samples over a period of 14-38 weeks. Based on comparable results in BM and PB samples and doubling times of 15-20 days, we propose that monitoring of AML patients should include PB sampling each 4-6 weeks. In one patient the relapse was not preceded by a gradual increase of double-positive cells. This false negative result was caused by a phenotypic shift, since at relapse the AML cells did not express TdT. In the five AML patients who still are in continuous cytomorphological CR for 32-46 months we repeatedly detected relatively high percentages of myeloid-marker+, TdT+ cells in BM (up to 0.1%) and PB (up to 0.02%). Although we could not prove the leukemic origin of these double-positive cells, they might represent residual dysplastic AML cells which survived chemotherapy but which are not capable of causing leukemia regrowth as yet. This would be in line with recent polymerase chain reaction studies, which could demonstrate the persistence of leukemic clones in the majority of AML patients in continuous CR. It is concluded that double immunofluorescence labeling for myeloid markers and TdT is useful for detection of residual disease in TdT+ AML patients. A gradual increase of double-positive cells is suggestive for leukemic cell growth and can be used to predict relapse.