In vitro induction of myelopoetic colonies from mouse bone marrow has been used for measurement of leucopoetic colony stimulating activity (CSA) isolated from large batches of human urine. After high flow dialysis in artificial kidneys and immediate adsorption to DEAE-Cellulose, followed by purification on Con A-Sepharose, treatment with insoluble Papain and gelfiltration on Sephadex G 100, enrichment of CSA was about 6,000-fold. An important step of the enrichment procedure was the separation from a CSA-inhibiting protein, probably combining with CSA. Specific activity was further increased by preparative polyacrylamide gel electrophoresis to 5.3 X 10(6) units per mg protein. The total enrichment exceeded 25,000-fold. The final purification product consisted of a group of closely related proteins with high specific activity. Antisera raised with one of the electrophoretic fractions suppressed bioactivity in each of the different purification steps including the final CSA fractions differing in electrophoretic mobility. The antisera furthermore inhibited CSA in human lung and monocyte conditioned media but had only very little effect on partially purified CSA from stimulated human lymphocytes as well as CSA derived from mouse lung conditioned medium.