Proliferation and differentiation of human granulocytes were studied in a diffusion chamber (DC) culture system. Evaluation of the nucleated marrow cells of normal volunteers revealed that the granulocytic precursors continue differentiation in the DC as they do in vivo. Generation time of the granulocytes in the multiplicative pool in DC was determined by the autoradiographic mean grain count halving method. With tritiated thymidine (3HTdR) the half-time was 67 h, where as with 125Iododeoxyuridine it was 47 h. The longer half-time with 3HTdR supports the notion that a fraction of nuclear 3HTdR from dead cells is reutilized by the newly proliferating cells. The 3HTdR labeling index of granulocytic precursors in the multiplicative pool during the first 10 days in DC is not constant, suggesting a variation in the rate of proliferation. The DNA synthesis time in DC, determined by double isotope labeling technique, showed that it was relatively constant. Simultaneous evaluation of rate of transition of granulocytes in DC from one stage to the next of one patient with chronic myelocytic leukemia and another with myeloid metaplasia with myelofibrosis indicated a more rapid transition in the DC than in vivo in man.