Objectives: To characterize a serological test for diagnosing endocarditis caused by Gram-positive cocci. Methods: We have developed an indirect enzyme-linked immunosorbent assay (ELISA) for the serological detection of Gram-positive infections. The test measures serum IgG directed towards lipid S, a recently identified exocellular glycolipid antigen which is related to lipoteichoic acid. We have previously shown the test to be of value in serodiagnosis of central venous catheter-associated sepsis and infection of orthopaedic prostheses caused by coagulase-negative staphylococci. We now describe the application of this test in endocarditis. Results: Serum IgG levels to lipid S were significantly elevated in 34 patients with Gram-positive bacterial endocarditis confirmed as 'definite' by the Duke criteria as compared to 50 control patients. The test had a sensitivity of 88% and a specificity of 88%. Conclusions: The assay is independent of culture results or endocardial imaging, making it complementary to currently used investigations. It may therefore be possible to refine the current Duke criteria for diagnosing endocarditis. We describe an algorithm which incorporates lipid S serology into a positive diagnostic strategy.