To improve the yield of the cytogenetic analysis in patients with CML at presentation and during alpha-interferon therapy, three culture conditions for bone marrow or peripheral blood cells were tested in parallel. The effects of 5637 conditioned medium (CM), nutritive elements (NE), and methotrexate (MTX) cell synchronization were investigated in 10 Ph-positive (Ph+) CML patients at diagnosis (group 1), and in 13 Ph+ CML patients receiving treatment with alpha-interferon (group 2). In the presence of 5637 CM and NE with or without MTX, the mitotic index values were significantly improved in both groups. In group 2, the morphological index was significantly increased when using 5637 NE, and percentages of abnormal cells did not differ in 5637 NE and 5637 NE MTX compared to the control condition. Although cessation of interferon administration before sampling may improve the yield of the technique, it does not seem necessary when using 5637 CM and NE. The variability of the response of leukemic cells to different culture conditions further supports the recommendation that, in addition to the control condition, supplementations with 5637 CM and NE with or without cell synchronization be used in parallel in all CML patients. Results suggest that, when the number of cells available is not sufficient for several cultures, 5637 NE with or without MTX should replace the control condition.