The aim of this study was to examine whether soluble neoepitopes of activated C3 (C3b, iC3b, C3c) and C9 are produced by human alveolar macrophages cultured in serum-free medium. There was a significant and inhibitable production of C3 and C9 neoepitopes and C9 by the macrophages from all donors, as detected by enzyme-linked immunosorbent assays based on monoclonal (bH6, aE11) and polyclonal (anti-C9) antibodies. A strong donor-dependent variation in the levels of the C3 neoepitope and C9 (five- to sevenfold) and the C9 neoepitope (twofold) was found. After 1 day (24 h) of incubation, the complement levels were largely unaltered. The presence of an exogenous alternative pathway activator (agarose beads) reduced the amount of soluble complement because of binding to the agarose. However, the relative fraction of C9 neoepitope versus C9 increased (two- to threefold), due to agarose-mediated activation of C9. The results demonstrate activation of the complement system in serum-free alveolar macrophage cultures, irrespective of the presence of a known complement activator.