A method that allows the routine cytogenetic analysis of colonies growing in semisolid medium and containing as few as 50 cells is described. This method is based on the sequential addition of fluorodeoxyuridine and then thymidine to achieve higher mitotic yields by the synchronization of colony cells. In this study the schedule outlined was specifically optimized for application to small granulopoietic colonies from patients with Philadelphia chromosome (Ph)-positive chronic myeloid leukemia. In addition to increasing the spectrum of colony sizes yielding analyzable metaphases, this method significantly improved the general quality of the chromosome preparations obtained. Minor changes should enable this method to be applied to the analysis of colonies derived from a variety of other normal or malignant clonogenic cell types.