Crude pyrogen was obtained by stimulating 1.4 x 10(12) peritoneal-exudate leukocytes from rabbits in vitro with Escherichia coli endotoxin. A pyrogen molecule was isolated by ammonium-sulfate precipitation, polyacrylamide gel electrophoresis at pH 5.5 and 8.9, and gel filtration on Bio-Gel P-100. These procedures yielded a pyrogenic fraction which was electrophoretically and immunologically homogeneous. The pyrogenicity was 50 ng/kg of rabbit. This leukocyte pyrogen consisted of a protein with a molecular weight of approximately 14 000. Amino-acid analysis showed high amounts of glutamic acid and glycine; methionine and tryptophan were present in small quantities. Arginine was found to be the N-terminal amino acid. The molecule contained about 0.8% neutral sugar and 0.7% lipids. Its approximate isoelectric point in citrate/phosphate buffer was 4.2. The activity was lost by lyophilization and storage at - 18 degrees C for six months. The data presented here are the first detailed characterization of a rabbit leukocyte pyrogen. The properties in common to leukocytic pyrogen and interleukin 1 are discussed.