Multiplication-stimulating activity, a family of insulin-like growth factors previously identified in medium conditioned by the BRL-3A rat liver cell line, is also synthesized by third passage cultures of rat embryo fibroblasts (REFs) maintained in serum-free medium. Conditioned serum-free medium from REFs was chromatographed on Sephadex G-75 in 1 m acetic acid, and fractions in the size range of BRL-MSA contained MSA immunoreactivity. A dose-response curve of pooled G-75 fractions was parallel to BRL-MSA standard, and levels in the REF-conditioned medium were 0.5-1 microgram/ml. REF-MSA from the Sephadex G-75 pool was equipotent to BRL-MSA in stimulating [3H]thymidine incorporation into DNA in chick embryo fibroblasts and in stimulating DNA synthesis in REFs, as measured by autoradiography. In receptor binding assays using REFs, chick embryo fibroblasts, Swarm rat chondrosarcoma cells, and rat liver membranes, REF MSA was equal to BRL MSA in competition for binding of [125I]iodo-MSA. REF MSA also behaved identically to BRL MSA in a competitive binding protein assay using binding proteins in rat serum. Further characterization of REF MSA using Bio-Gel P-10 chromatography, followed by high pressure liquid chromatography or polyacrylamide gel electrophoresis, indicated that immunoreactive polypeptides in the fibroblast medium corresponded to BRL MSA II (mol wt, 8700) and BRL MSA III (mol wt, 7100). The amount of REF MSA released into the medium increased linearly over time. Cycloheximide decreased the amount of MSA in the medium, and during a recovery period, the amount of MSA returned nearly to control levels. In summary, rat embryo fibroblasts synthesize MSA which is biologically, immunologically, and chemically identical to MSA produced by the BRL-3A rat liver cell line.