Sequential changes in the distribution of lymphocyte subpopulations of the thymus and spleen in BALB/c mice (male, 8-weeks old) during 2 weeks after a single i.p, injection of cyclophosphamide (CY, 200 mg kg body weight) were studied mainly through the use of CD4, CD8 and CD3-E markers together with single-or two-color flow cytofluorometry. In the thymus on Day 2 after CY treatment, a marked decrease in the number and proportion of PNAh', CD3-and CD4+CD8+ double positive (DP) subpopulation was observed in parallel with a marked reduction in the thymus weight, cortical area and total thymocyte number. This phenomenon might be associated with the decrease in the percentage of thymocytes at the S phase of the cell cycle on Day 1 and 2 after CY treatment, owing to the depletion of the rapidly dividing cells in DP subset. There was a significant reduction in the number and proportion in CD4+ single positive(SP) subset on Day 7. The cell number of CD8+SP subset continued to decrease during Day 7 to Day 14. This contrasted with the behaviors of DP, CD4+SP and CD4-CD8-double negative (DN) subsets in which a considerable recovery was attained by Day 14. The spleen from CY-treated mice showed a marked decrease in the DN subset and surface immunoglobulin-positive cells, perhaps B lymphocytes, in both the percentage and the absolute cell number on Days 2 and 7, which paralleled the marked reduction in its weight and total cell number. The absolute cell numbers of CD4+SP and CD8+SP subsets in the spleen were also reduced on Days 2 and 7. The reduction of the CD4+SP/CD8+SP ratio was found in the thymocytes on Days 2 and 7 but not in spleen cells. Our results suggest that the principal target cell population of CY is the PNAh', CD3-and DP immature cortical thymocytes as well as splenic B cells. The sustained decrease in the number of CD8+SP thymocytes after CY treatment might be in part relevant to the potentiating effect of CY pretreatment on immune responses.