Continued studies of the macrophage-derived mediator of SAA synthesis (SAA Stimulating Factor) confirm our previous observations that SAASF copurified with leukocytic pyrogen (LP) and lymphocyte activating factor (LAF). Moreover, new data demonstrate three separate isoelectric points for human LP-LAF-SAASF each of which possess the three biological activities. During the purification of 15,000 MW LP from crude stimulated mononuclear cell supernatants, only those fractions with pyrogenic activity in rabbits caused augmented stimulation of lymphocytes (LAF) and induced SAA synthesis in mice. Purified human LP stimulated isolated mouse hepatocytes in vitro to synthesize SAA in a dose-responsive manner. Colchicine treatment of hepatocytes led to decreased secretion of SAA into the medium and to an intracellular accumulation of SAA. Messenger RNA was isolated from the livers of endotoxin-stimulated mice and translated in a wheat-germ cell-free system. A major product was identified at 13-14,000 MW. Immunoprecipitation with anti-mouse AA identified several bands on autoradiography of polyacrylamide gels. These larger SAA precursors may account for the previously noted heterogeneity of human SAA, comprising at least 6 SAA isomers, of similar molecular weight but different solubility and electrophoretic charge characteristics. Two monoclonal antibodies (IgM-K and IgG1-K) have been prepared using standard cell hybridization techniques. They are directed at the variable COOH terminal region of SAA since they detect differences between the 6 human SAAs but do not react with human, monkey, dog or mouse AA proteins, human AP, C-reactive protein, IgG nor albumin. These antibodies will be useful in examining the origin, structure and function of SAA.