The replication status of malignant cells is usually asynchronous. However, to date the pattern of replication has not been studied in myeloproliferative disorders nor has the effect of chemotherapy been systematically evaluated. Therefore, we used fluorescence in situ hybridization to interphase nuclei in PHA-stimulated peripheral blood lymphocytes to examine replication timing of three alleles associated with the malignant process. The study group comprised hydroxyurea treated and untreated patients with essential thrombocytosis (ET) or polycythemia vera (PV). A significantly higher rate of the asynchronous pattern of replication in both treated and untreated patients was found as compared to healthy controls. The highest rate of asynchronous replication was observed in untreated patients. Also, the frequency of the two doublets pattern was significantly higher in the untreated group compared to the treated patients and to the control groups. In conclusion, patients with PV and ET have a higher rate of asynchronous pattern of replication. A possible correlation between disease activity and the pattern of replication is suggested. The effect of hydroxyurea on the pattern of replication is variable.