Resident, elicited, and activated mouse peritoneal macrophages exhibit a differential expression of the activity of the enzyme b̃‐galactosidase; freshly harvested resident macrophages express a remarkably high activity whereas the latter two populations are almost void of enzymic activity. During in vitro cultivation there is an enhancement in the level of the enzyme in the three populations, and a significant proportion of both thioglycollate‐elicited and Corynebacterium parvum‐activated macrophages acquire b̃‐galactosidase activity. Cells within in vitro differentiated bone marrow‐derived mononuclear phagocyte colonies are heterogeneous with respect to expression of b̃‐galactosidase activity. The percentage of cells expressing medium to intense enzymic activity is augmented with time in culture. Essentially the same pattern is observed in colonies differentiated from bone marrow of mice bearing acute or chronic inflammation. Freshly isolated human peripheral blood monocytes are essentially void of detectable b̃‐galactosidase activity. Eighty to ninety percent of the monocytes acquire medium to intense activity during a 7‐day cultivation period. The data support the suggestion that b̃‐galactosidase expression in mononuclear phagocytes is a correlate of their maturational stage both in vivo and in vitro and does not reflect the state of elicitation or activation of these cells.