The serum lipopolysaccharide (LPS) binding protein, LBP, has been shown to greatly enhance cellular responses to low concentrations of LPS. Purified LBP facilitates the transfer of LPS to membrane-bound or soluble CD14; the CD14/LPS complex then triggers a signal in responsive cells. We have cloned and sequenced a cDNA encoding murine LBP, and produced recombinant murine LBP using a baculovirus expression system. Using either a solid-phase or a cytofluorometric assay, recombinant murine and human LBP were found to bind avidly to free LPS, but only weakly to live bacteria from most LPS-containing Gram negative strains. Binding correlated loosely with the length and composition of the polysaccharide O chains. However, recombinant LBP did bind well to all heat-killed bacterial preparations. These findings suggest that LBP could be implicated in the response to killed but not live Gram negative bacteria.