Anticardiolipin antibodies (ACA) were detected by solid-phase enzyme immunoassay in the majority of sera from patients with Gram-positive and Gram-negative bacterial infections. The response involved all the major immunoglobulin classes IgG, IgM, and IgA. The specificity of the ACA was studied in competitive inhibition experiments with three putative antigens: cardiolipin, lipopolysaccharide (LPS) isolated from Salmonella minnesota, strain Re 595, and synthetic Escherichia coli lipid A. The binding of IgG class ACA from the sera of five patients with Gram-negative infections was effectively inhibited by LPS, whereas 100-fold more cardiolipin was required for comparable inhibition. Pure lipid A was a less effective inhibitor of anticardiolipin activity than LPS. This pattern of reactivity was not seen in sera from patients with Gram-positive infections, syphilis, or systemic lupus erythematosus. Our findings suggest that cardiolipin may not be the inducing antigen for the cardiolipin-binding antibodies that develop in Gram-negative infections.