Laboratory testing for B. burgdorferi infection is intended to substantiate a physician's clinical judgment of whether a patient has Lyme disease or not. Cultivation of B. burgdorferi from a patient's skin or blood is the gold standard for demonstration of active infection, but it is expensive and lacks clinical sensitivity. Detection of spirochetal DNA in clinical samples by PCR has better sensitivity, but PCR for B. burgdorferi has not yet been standardized for more routine diagnostic testing. Detection of antibodies to B. burgdorferi is the most practical and common approach for laboratory work-up of a case of suspected Lyme disease. Serologic assays fall short of 100% sensitivity and specificity, however, and examination of a single specimen in time does not discriminate between previous and ongoing infection. Because of a background false positivity even among healthy populations of nonendemic regions, serologic testing is recommended only when there is at least a one in five chance, in the physician's estimation, that the patient has active Lyme disease. The pretest likelihood of the disease is determined by the physician in the context of epidemiologic and clinical facts of the case. This estimate can serve to reassure patients who are at low risk of B. burgdorferi infection but are seeking a Lyme test for complaints of a more nonspecific nature. Although new subunit serologic assays based on recombinant proteins are becoming available commercially, the longstanding two-test approach, in which a positive or indeterminate result with a standardized, sensitive ELISA test is followed by verification with a more specific Western blot assay, still provides the physician with a reasonably accurate and reliable assessment of the presence of antibodies to B. burgdorferi. More recent challenges for serologic testing are seropositivity in the population as the result of immunization with the Lyme disease vaccine and the emergence of new Borrelia species that cause Lyme disease-like illnesses.