Pro-inflammatory cytokines attract leukocytes to inflamed tissues and activate them. Few attempts have been made to identify the sources of cytokines in vivo. We examined the importance of peritoneal macrophages in the mobilization and homing of neutrophils to a sterile peritonitis in the rat, with emphasis on their cytokine production. Macrophages, present in virtually all tissues, are known to be easily activated and to serve as an important source of cytokines. Flow cytometric analysis of cells stained intracellularly with tagged antibodies against various cytokines revealed that the peritoneal macrophages were stimulated to produce the following cytokines: interleukin (IL)-1beta, macrophage inflammatory protein-2 (MIP-2), and keratinocyte-derived cytokine (KC). High numbers of neutrophils, activated on arrival into the peritoneal cavity, also produced IL-1beta, whereas lower numbers contained interleukin-6, tumor necrosis factor-alpha, MIP-2, KC, and MIP-1alpha. This marked activation of peritoneal neutrophils was also reflected by increased surface expression of CD11b. On the other hand, peritoneal macrophages expressed high basal levels of CD11b, which were reduced 24 h after the onset of inflammation. In rats selectively depleted of macrophages by i.p. injection of liposome-containing clodronate, the massive influx of neutrophils to the peritoneal cavity was markedly reduced, as was the rapid mobilization of mature bone marrow neutrophils. Local macrophages are important both for the accumulation of neutrophils in the inflamed peritoneal cavity and for the early mobilization of neutrophils from the bone marrow. Macrophage-derived IL-1beta, MIP-2, and KC are possible mediators of neutrophil homing to inflamed tissues.