In an attempt to define the relation among anemia, tumor mass, and proliferative activity of tumor cells in vivo, we measured the proportion and cell cycle distribution of erythropoietic cells and myeloma cells in the bone marrow of patients with multiple myeloma using four-parameter flow cytometry. Forty-three bone marrow samples from 33 patients with stage II or III disease and normal renal function at diagnosis (n = 9), in partial remission (n = 9), and in progression or relapse after chemotherapy (n = 25) were evaluated. Early and late erythropoietic cells were discriminated based on published light scatter properties in combination with CD71 expression. Myeloma cells were detected by exploiting their strong CD38 positivity and light scatter characteristics. Cell cycle distribution of the three cell populations was determined by propidium iodine staining. In the whole group of patients, hemoglobin (Hb) concentration was inversely correlated with beta2-microglob-ulin (p = 0.03), percentage of marrow CD38++ cells (p = 0.008), and percentage of CD38(++) cells in S phase (S-CD38++; p < 0.001). Partial correlation analysis revealed S-CD38++ to be the only independent predictor of Hb concentration (p < 0.001). No correlation was found between Hb concentration and the S-phase fraction of erythropoietic cells. In the subgroup of patients with moderate to severe anemia, defined as Hb concentration <11 g/dL, Hb level correlated negatively only with S-CD38++ (p < 0.001) but not with beta2-microglobulin and percentage of marrow CD38++ cells. In addition, Hb and the S-phase proportion of early erythropoietic cells correlated positively (p = 0.029). The strong inverse correlation between Hb concentration and percentage of myeloma cells in S phase suggests that in multiple myeloma, tumor proliferative activity may have a more important impact on the development of anemia than tumor mass. The S-phase fraction of tumor cells appears to be the most important pathogenic factor, especially in anemic patients. In these patients, the positive relation between Hb concentration and the S-phase fraction of erythropoietic progenitors indicates that development of anemia is associated with inhibition of erythropoiesis.