Macrophage populations from human fetal liver were examined for the sequential appearance of different antigenic determinant during maturation. Frozen sections of liver, from 12 to 21 weeks gestation were analyzed using a series of four monoclonal antibodies with known specificity. The macrophage monoclonal antibodies used were OKM-1, which defines monocytes, macrophages, and granulocytes; Leu M-3 and MO-2, which identify monocytes and macrophages; and 6B8, a new macrophage monoclonal antibody which binds to tissue macrophages. The staining pattern described by each of these monoclonal reagents was compared with the distribution of morphologically distinguishable tissue macrophages in fetal liver, based on the expression of surface and/or cytoplasmic antigens. The data indicate that the antigens defined by OKM-1 and 6B8 are present on large numbers of cells as early as 12 weeks gestation. In contrast, the antigenic determinants identified by Leu M-3 and MO-2 are present only on cells in 15 to 21 weeks of gestation; thus these antigens are mature differentiation antigens. Furthermore, double-staining studies confirmed that with the increase in fetal age unique macrophage populations can be identified based on the matrix of antigenic determinants. Thus, macrophage heterogeneity in the fetal liver may be a function of maturation.