Ascites sarcoma 180 (S180A) is a transplantable tumor which causes hypercalcemia in tumor-bearing mice, and stimulates bone resorption without parathyroid hormone-like activity. In the present study, parathyroid hormone-related protein (PTHrP) mRNA could not be detected in total RNA from S180A cells. Bone-resorbing activity (BRA) derived from serum-free conditioned medium of S180A cells (S180A-CM) was coeluted with either transforming growth factor alpha (TGF alpha) activity (peak A, approximate M(r), 29 kDa) or lymphocyte-activating factor (LAF) activity (peak B, M(r), 20.1-24 kDa) in Bio-Gel P-100 column chromatography. Fractions in peak A and B contained IL-6 but not tumor necrosis factor alpha (TNF alpha). Subsequent separation of peak A by reverse-phase high performance liquid chromatography produced a single fraction which contained both BRA and TGF alpha activity. Recombinant human TGF alpha-induced bone resorption was completely inhibited by indomethacin. The BRA in peak A was partially inhibited by indomethacin and almost completely inhibited by simultaneous treatment of indomethacin and anti-IL-6 antibody. The BRA in peak B was partially inhibited by neutralization with anti-IL-1 alpha antibody and was completely inhibited by simultaneous treatment with anti-IL-1 alpha and anti-IL-6 antibody in the absence of indomethacin. Bone resorption induced by S180A-CM was associated with an increased production of prostaglandin E2 (PGE2) by calvaria. The BRA in S180A-CM, however, was not completely abolished by the simultaneous addition of indomethacin and anti-IL-1 alpha, anti-IL-1 beta and anti-IL-6 antibodies. Our findings indicate that (1) BRA derived from S180A cells includes TGF alpha, IL-1 alpha, IL-6 and some other unknown factor(s), distinct from PTHrP, IL-1 beta and TNF alpha, and (2) these unknown factors resorb bone in part via a PGE2-independent pathway.