We investigated the time-course involved in the conversion of mouse blood monocytes in vitro to cells capable of anchorage-independent growth. Two criteria were used to define when monocytes were fully converted to cells similar to mononuclear phagocytes present in inflammatory exudate, such as thioglycollate medium (TM)-elicited peritoneal exudate. They were the production of high levels of plasminogen activators and an ability to undergo anchorage-independent growth. Resident peritoneal macrophages were used as controls and for comparison. Our studies indicated that monocytes, but not resident peritoneal macrophages, could be converted to cells similar to TM-elicited mononuclear phagocytes after 2 d in culture.