Methotrexate-thymidine synchronization increased mitotic yield and the numbers of cells with longer chromosomes when compared with direct and day culture (24 hr) techniques. The longer chromosomes overlapped more than shorter ones, but this adverse effect of cell synchronization was outweighed by the substantial gains from increased band number of metaphase cells. A critical feature of the synchronization technique that determines chromosome length is the period of cell culture following thymidine release. This will depend on cell cycle time. Variable results obtained with the synchronization technique probably occur because the cell cycle time of leukemic cells differs from that of normal hematologic cells and normal lymphocytes. It may also differ between patients and between acute and chronic forms of leukemia.