A method has been developed for preparation of confluent monolayers of human monocytes from small volumes of blood and for maintenance of these pure monocyte cultures for up to 16 wk in vitro. These cells phagocytosed 5.7 mum diameter latex beads, rosetted with erythrocytes coated with IgG or with C3, killed Listeria monocytogenes, and synthesized both lysozyme and the second component of complement. Lysozyme was secreted at a rate of approximately 50,000 mol/min per cell for at least 12 wk in cultures. The maximal rate of C2 synthesis and secretion was considerably less; i.e., approximately 30 mol/min per cell between the 2nd and 12th wk in culture. Monocytes produced little C2 during the first 6 days in culture after which a marked increase in the rate of C2 production was noted. This increase was coincident with morphologic evidence of monocyte maturation.