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BACKGROUND: Although rapid diagnosis of Lyme disease is essential for effective treatment, there is concern about inappropriate testing. We conducted a prospective, cross-sectional survey of clinicians to assess the use and appropriateness of Lyme disease serologic tests (LDSTs). METHODS: LDSTs performed at 2 large Wisconsin reference laboratories were systematically sampled for 12 consecutive months. A standardized questionnaire was used to gather data about the submitting clinician and the patient tested. Tests were categorized as appropriate, inappropriate, or discretionary, and associations were assessed using logistic regression analysis. A test was defined as inappropriate if the patient was asymptomatic, had erythema migrans, or was treated empirically, or if the test was ordered as a test of cure. RESULTS: We surveyed 303 clinicians regarding 356 LDSTs: 72 tests (20%) were appropriate, 95 (27%) were inappropriate, and 189 (53%) were discretionary. Tests were more likely to be inappropriate if they were ordered by an emergency or urgent care physician compared with other specialists (adjusted odds ratio [AOR] 5.2, 95% confidence interval [CI], 1.3-20.6), or if preceded by a known tick bite (AOR 6.8, 95% CI, 2.6-17.6). The patient rather than the clinician requested 26% of tests, which were more likely to be inappropriate than clinician-requested tests (crude odds ratio [COR] 5.8, 95% CI, 2.5-13.6). Tests were more likely to be patient-requested if they were ordered by an internist (AOR 2.6, 95% CI, 1.4-4.8) or if the patient was > or =40 years old (AOR 2.2, 95% CI, 1.3-3.9). CONCLUSIONS: Many LDSTs are ordered inappropriately, often influenced by patient demand. Education of clinicians and patients about testing indications and contraindications is needed to reduce the number of inappropriate LDSTs.