Human cultured monocyte-like tumor cells of the U-937 line, that are incubated for 24 h at 37 degrees C with 100 ng/ml of PMA, generate and release a fibroblast-activating factor (FAF) capable of enhancing the uptake of [3H]TdR by human dermal fibroblasts in vitro. The predominant FAF activity in unpurified supernatants from U-937 cells was associated with one protein of 16 to 18 kDa, as assessed by SDS-PAGE and pI 4 to 5, as determined by IEF in gel. Isolation of the FAF in 15-liter batches of supernatant from cultures of 1.5 x 10(10) PMA-stimulated U-937 cells by ammonium sulfate precipitation and sequential filtration on Sephadex G-50, anion exchange chromatography, and reversed phase HPLC yielded microgram quantities of a homogeneous protein of sufficient purity for structural studies. Purified FAF was not absorbed by affinity columns bearing antisera to IL-1 beta, TNF or basic fibroblast growth factor die not share any tryptic peptides with IL-1 alpha or acidic fibroblast growth factor when analyzed by two-dimensional electrophoresis/chromatography on cellulose thin-layer plates, and has an amino-terminal sequence of amino acids that is different from any known fibroblast growth factor. FAF thus represents a unique human monocyte-derived protein that selectively stimulates human fibroblast proliferation and other functions.