Macrophage activation is associated with the production and release of reactive oxygen species (ROS), which are capable of mediating oxidative modification of low-density lipoprotein (LDL). In the present study we questioned whether cellular capacity to oxidize LDL increases during in vivo monocyte/macrophage maturation. We developed a novel model for macrophage maturation in vivo using mouse peritoneal macrophages (MPMs) harvested at increasing intervals after intraperitoneal thioglycollate injection. Macrophage maturation was evidenced by a progressive increase in cellular size, density, granulation, and expression of cell surface markers CD11b and CD36, and by a gradual decrement in myeloperoxidase activity. Cellular capacity to stimulate copper ion-mediated oxidation of LDL increased gradually by up to 2-fold during in vivo macrophage maturation in Balb/C mice, similar to the pattern observed during 1,25-dihydroxyvitamin D3-induced in vitro differentiation of the PLB-985 cell line. These effects were attributed to a gradual increase in production of ROS by up to 9-fold. The mechanism for the increase in cellular oxidative stress during macrophage maturation could be related, at least in part, to NADPH oxidase activation, as demonstrated by a gradual increase over time in p47phox expression (mRNA and protein) and in its translocation to the plasma membrane. In conclusion, in vivo monocyte-to-macrophage differentiation is associated with increased cell capacity to oxidize LDL, which may represent a protective mechanism for rapid removal of atherogenic LDL from extracellular spaces in the arterial wall.