The effect of leukoregulin, a 50-kD lymphokine with unique antitumor properties, was studied in vitro on several fibroblast functions. Leukoregulin did not inhibit fibroblast proliferation, as measured by cell enumeration and [3H]thymidine incorporation, and had no cytotoxic effect in terms of increased membrane permeability detected by trypan blue exclusion, two of the major leukoregulin actions on tumor cells. Leukoregulin induced a dose-dependent decrease in collagen synthesis, demonstrated by decreased [3H]proline incorporation into collagenase- digestible protein, as early as 6 h after the addition of the lymphokine to human fibroblasts. Leukoregulin inhibited the synthesis of both type I and type III collagen, as measured by SDS-PAGE and by specific radioimmunoassay. Neutralizing antibodies to interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, and interferon- gamma failed to alter the effect of leukoregulin on collagen synthesis, attesting that leukoregulin action was not due to contamination by these cytokines. Inhibition of collagen synthesis occurred concomitantly with increased secretion of prostaglandin E2 and a transient rise in intracellular cyclic AMP content, peaking at 6 h. However, blocking prostaglandin synthesis with indomethacin did not counteract inhibition of collagen synthesis by leukoregulin, demonstrating independence of this action of leukoregulin from cyclooxygenase metabolites. Leukoregulin also stimulated glycosaminoglycan production in a dose-dependent manner, affecting the synthesis of hyaluronic acid as the major fibroblast-derived extracellular glycosaminoglycan. In addition, secretion of neutral proteases (collagenase, elastase, caseinase) was increased. These observations indicate that leukoregulin is able to regulate synthesis of molecules critical to the deposition of the extracellular matrix by nontransformed nonmalignant fibroblasts.