Hemolin, a plasma protein from lepidopteran insects, is composed of four immunoglobulin domains. Its synthesis is induced by microbial challenge. We investigated the biological functions of hemolin in Manduca sexta. It was found to bind to the surface of bacteria and yeast, and caused these micro-organisms to aggregate. Hemolin was demonstrated to bind to lipopolysaccharide (LPS) from Gram-negative bacteria and to lipoteichoic acid from Gram-positive bacteria. Binding of hemolin to smooth-type forms of LPS was competed for efficiently by lipoteichoic acid and by rough mutant (Ra and Rc) forms of LPS, which differ in polysaccharide length. Binding of hemolin to LPS was partially inhibited by calcium and phosphate. Hemolin bound to the lipid A component of LPS, and this binding was completely blocked by free phosphate. Our results suggest that hemolin has two binding sites for LPS, one that interacts with the phosphate groups of lipid A and one that interacts with the O-specific antigen and the outer-core carbohydrates of LPS. The binding properties of M. sexta hemolin suggest that it functions as a pattern-recognition protein with broad specificity in the defense against micro-organisms.