Bloodstream invasion is an important event in the pathogenesis of the more serious manifestations of Lyme disease. The number of spirochetes in the blood of infected patients, however, has not been determined, and, therefore, it is unknown whether the number of spirochetes can be correlated with particular clinical or laboratory features. This study was designed to measure the level of Borrelia burgdorferi in the plasma of Lyme disease patients and correlate these levels with selected clinical and laboratory findings. Nested and quantitative polymerase chain reaction (qPCR) was employed to detect cell-associated flaB gene DNA in the plasma of untreated early Lyme disease patients with erythema migrans (EM). Twenty-nine (45.3%) of 64 patients had evidence of B. burgdorferi in their plasma by at least one of the PCR methods. For the 22 qPCR-positive patients, the mean number of flaB gene copies per mL of plasma was 4,660, with a range of 414 to 56,000. The number of flaB gene copies did not significantly correlate with any of the clinical, demographic, or laboratory variables assessed. For reasons discussed, we suggest caution in extrapolating an estimate of the number of viable Borrelia in plasma from the observed number of flaB copies.