The role of macrophages in neovascularization of tumors was investigated by examining the ability of tumor-associated macrophages (TAM) and their conditioned culture media to induce neovascularization in the cornea of syngeneic rats and proliferation of bovine aortic endothelial cells in culture. TAM were isolated from a 3-methycholanthrene-induced fibrosarcoma propagated in F344 male rats by enzymatic dissociation and were purified by centrifugation through continuous Percoll density gradients, followed by adherence to fibronectin-coated dermal collagen gels. The angiogenic potential of (a) TAM and their 72-hour conditioned culture media, (b) whole tumor cell suspensions (WTCS), (c) tumor cell suspensions depleted of TAM (TCS), and (d) macrophage-depleted tumor cell suspensions reconstituted with TAM (TCS + TAM) were compared. Cells were injected directly; conditioned media were concentrated 10-fold, incorporated into slow-release Hydron pellets, and implanted intracorneally. Stimulation of bovine aortic endothelial cell growth by TAM was assayed in culture with TAM-conditioned media and compared with responses induced by conditioned media from peptone-elicited rat peritoneal exudate macrophages. TAM and their conditioned media induced neovascularization in 38 of 40 corneas (95%) and 15 of 17 corneas (88%), respectively. Maximal vessel ingrowth occurred by the 5th day of implantation. Neovascular responses induced by WTCS (24 of 26 corneas, 92%) and TCS (17 of 24 corneas, 71%) occurred on the 7th and 10th day, respectively. TCS + TAM induced neovascular responses comparable to those elicited by WTCS (19 of 20 corneas, 95%). Addition of TAM-conditioned media to bovine aortic endothelial cell cultures stimulated a 10-fold increase in cell number within 10 days. This growth stimulatory effect was comparable to or greater than responses induced by conditioned media from rat peritoneal macrophages. Our results demonstrate that TAM are potent stimulators of neovascularization and endothelial cell proliferation and that depletion of macrophages from tumor cell suspensions significantly decreased their angiogenic potential. This suggests that neovascularization of this tumor is mediated in part by macrophages.