The effects of sera containing in vivo activated cyclophosphamide were tested on macrophage granulocyte colony growth (CFUc) and mixed leukocyte cultures (MLC). Bone marrow reconstitution and the growth of a canine tumor after incubation of transplanted cells with active sera were studied in vivo. The following experiments were performed: 1) peripheral blood CFUc values were determined in 6 dogs 5 min to 72 h after intravenous administration of 100 mg/kg cyclophosphamide. Inhibition was apparent at 5 min and lasted for 24 h. 2) Sera samples were obtained from 10 dogs after cyclophosphamide administration. The half life of serum inhibitory activity measured against normal dog cells was approximately 6 h. Inhibitory effects were noted 5 min following drug administration and complete at 30 min. 3) Timed incubation with 1 h postcyclophosphamide sera revealed preferential inhibition of MLC activity compared to CFUc activity, 4) In vitro incubation for 30 min in active sera did not affect the bone marrow repopulating potential of hematopoietic cells compared to nonincubated cells. 5) At least a tenfold reduction in the growth potential of a transplantable canine tumor could be produced by in vitro incubation of tumor cells with active sera. It is concluded that varying sensitivities of hematopoietic, immunocompetent and tumor cell populations to cyclophosphamide metabolites may provide a basis for in vitro chemoseparation of mixtures of these cell types.