Amyloid-enhancing factor (AEF) was extracted from spleens of mice that had received amyloidogenic stimulation. Sephacryl S-300 gel filtration of the crude AEF yielded five fractions, among which strong AEF activity was present in the first peak (F1), and confirmed by an amyloid induction experiment. An anti-AEF antiserum was obtained from a rabbit by immunization with F1. This antibody reacted strongly with splenic polymorphonuclear leukocytes (PML) from mice given amyloidogenic stimulation, and weakly with those from normal untreated mice. Isoelectric focusing (IEF) analysis of both F1 and sera from mice given amyloidogenic stimulation was performed. A single band was observed on IEF analysis of F1, whereas many bands were seen on IEF analysis of the sera. After the substances in the gel had been transferred to nitrocellulose membranes by capillary blotting, the membranes were made to react with the anti-AEF antiserum. The results suggested that AEF is a high-molecular-weight substance derived from PML and increases in the serum at the time of, or shortly prior to, amyloid deposition in the spleen.