The composition and characteristics of the bone marrow extracellular fluid supposedly modify the transport of cytokines, drugs, and other signaling molecules involved in the regulation of bone marrow function. Direct access to the bone marrow extracellular fluid surrounding hematopoietic cells is complicated by the virtually noncompliant surrounding bone tissue. We examined the applicability of a centrifugation method to obtain representative samples of bone marrow extracellular fluid from rats and humans. Perforated rat bones or human bone marrow biopsies were wrapped in nylon mesh baskets before being centrifuged at 180-239 g. In the rats, we found an only minor contribution of fluid from other sources than the bone marrow extracellular fluid as indicated by the average ratio of centrifugate-to-plasma activity of the extracellular tracer fluid 51Cr-labeled EDTA of 0.85. The colloid osmotic pressure in the centrifugate was consistently lower than that in the corresponding plasma in both species. In rats and humans, high-performance liquid chromatography showed a protein elution pattern from the bone marrow fluid similar to that of plasma, except for a peak eluting in the approximately 40-kDa molecular mass range. Western blotting of the cytokines erythropoietin and granulocyte colony-stimulating factor revealed generally higher amounts in the centrifugate than in the plasma. This difference was augmented during increased hematopoietic activity induced by inflammation or bleeding in rats. We conclude that the centrifugation method provides representative samples of bone marrow extracellular fluid and that extracellular signaling responses to altered hematopoiesis are more clearly reflected locally in the bone marrow interstitium than in plasma.