Ex vivo expansion systems of hematopoietic progenitor cells (HPC) have been extensively studied and their clinical application is under investigation. However, it is not known whether HPC expanded ex vivo will be able to retain their replating potential. CD34+ cells isolated from cord blood were cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 1.0% bovine serum albumin, 50 ng/ml stem cell factor, 50 ng/ml interleukin-3 (IL-3), 50 ng/ml IL-6, 100 ng/ml granulocyte colony-stimulating factor, and 3 U/ml erythropoietin for 0, 5, 7, 10, 14, and 21 days. After the expansion cultures, granulocyte/macrophage progenitor cells (CFU-GM) were assayed from each culture by the standard methylcellulose method. After 14 days of culture, CFU-GM-derived colonies were randomly picked up and processed for the replating assay. The fold increase of CFU-GM peaked at day 7 of the expansion culture (29.8 +/- 7.7-fold, n = 5), followed by a decline until day 21. In the replating assay of CFU-GM from freshly isolated CD34+ cells, the mean replating efficiency was 91.2 +/- 4.7%. The replating efficiency decreased gradually with the time of the expansion culture. At day 7 when the fold increase of CFU-GM reached its peak, the replating efficiency had dropped to 47.5 +/- 2.3%, followed by a further decline to 5.3 +/- 3.4% at day 21. Furthermore, the addition of 100 ng/ml thrombopoietin to this expansion system failed to prevent the decline of replating efficiency. These observations suggest that the replating potential of CFU-GM may decrease in the ex vivo expansion system, even when their fold increase reaches its peak. This should be taken into consideration when HPC expanded ex vivo are used in clinical transplantation.