Earliest origins of macrophage populations in the central nervous system, the liver, and the lungs were studied in rat embryos aged between 10.5-11 days and 14 days of gestation, based on light and electron microscopic identification of macrophages using peroxidase-coupled isolectin B4 of Griffonia simplicifolia (GSA I-B4), which recognizes alpha-D-galactose groups on the cell membrane. During embryonic life macrophages and their precursors are GSA I-B4-positive and generally bereft of peroxidase-positive granules. At 10.5 days the yolk sac and embryonic circulations have just become joined, the brain has five vesicles but nerve cells are little differentiated, the liver exists as a diverticulum of the gut with fingerlike extensions of hepatocytes, and the lungs as a laryngotracheal groove. Macrophages and/or their precursors occurred in small numbers in embryonic mesenchyme and blood vessels but showed no special affinity for either liver or lung rudiments. The developing brain was the first organ to be colonized, beginning on prenatal day 12. The liver followed between days 12 and 13 and was succeeded by the lungs, beginning between days 13 and 14. Dividing macrophages were present in these organs at the outset of colonization and throughout the duration of the embryo series, indicating that from the beginning, replication of resident cells contributes to growth of the local population. Granulocyte precursors were first apparent in the liver around day 13; they are also GSA-positive but are distinguished from macrophages by their content of peroxidase-positive granules. Organ cultures of 13-day liver and lungs, and 14-day brain tissue, indicate that whereas isolated liver fragments support the formation of both granulocytes and macrophages, only the latter develop in brain or lung cultures. A resident population of macrophages evidently is set up very early in these organs, well before white cells colonize the spleen, bone marrow, and other future blood forming regions. The events outlined are seen as stages in an embryo-wide process that leads to establishment of macrophage populations in various organs.