Fibroblasts migrate in vitro in response to a variety of chemoattractants including cellular, humoral, and connective tissue components. This report describes a low molecular weight, macrophage-derived chemotactic substance for fibroblasts (MDCF-F). Thioglycolate-elicited peritoneal exudate cells were collected from mice 4 days postinjection. Platelets were removed and glass-adherent macrophages were selected in the absence of fetal calf serum. The macrophage culture medium was then tested for chemotactic activity for 3T3 mouse fibroblasts using a modified Boyden chamber. Chemotactic activity was observed in the macrophage medium after a 48-hr culture period and increased during the next 24 hr. The activity was sensitive to heat treatment at 56 degrees C for 30 min, could be effectively absorbed out of the culture medium by preincubation with 3T3 fibroblasts, and was not recognized by human fibroblasts. Molecular-sieve chromatography indicated a molecular weight below 10,000. This low molecular weight, macrophage-derived chemoattractant for fibroblasts may represent a rapidly diffusable substance involved in the recruitment of fibroblasts to sites of inflammation.