Laboratory tests have been used extensively to help diagnose Borrelia burgdorferi infections. In many cases, results of indirect fluorescent antibody (IFA) staining methods or an enzyme-linked immunosorbent assay (ELISA), combined or separate from findings of Western blot analyses, have confirmed clinical diagnoses of Lyme disease. Alternative assays, such as culturing or DNA detection by polymerase chain reaction (PCR) methods, can provide more definitive evidence of B. burgdorferi infection than can antibody assays. However, aside from being more expensive, culturing B. burgdorferi from human tissues and fluids gives us a low yield, while results of PCR analyses can be as misleading as those obtained by performing IFA staining methods or an ELISA if there are false-negative or false-positive reactions. With increased knowledge of human immune responses to key proteins of B. burgdorferi, such as those with molecular masses of 21, 31, 34, 39, 41, and 93 kilodaltons, Western blot analyses are being used more frequently to confirm B. burgdorferi infections. These methods have been particularly helpful in identifying false-positive reactions in an ELISA. Until highly sensitive and specific assays have been adequately standardized, diagnosis of Lyme disease should be based primarily on clinical and epidemiologic evidence.