Monocytic cells exposed to Borrelia burgdorferi, through unknown receptors, overexpress the urokinase receptor (uPAR), a key mediator of the plasminogen activation system. We show that combined blockade of CD14 and TLR2 causes a significant inhibition of B. burgdorferi-induced uPAR in Mono Mac 6 (MM6) cells. Other pattern recognition receptors tested (CD11b/CD18, the mannose receptor, and the N-formyl-methionyl-leucyl-phenylalanine receptor) did not have demonstrated roles in B. burgdorferi-mediated uPAR induction. We dissected the result for CD14 andTLR2 by investigating the singular contributions of each. Independent functional blockade of CD14 or TLR2 failed to inhibit B. burgdorferi-mediated uPAR induction. 1,25-Dihydroxyvitamin D(3) differentiation of MM6 cells increased CD14 expression 12-fold but did not augment B. burgdorferi-mediated uPAR expression. Peritoneal exudate macrophages (PEM) from CD14- or TLR2-deficient mice were not defective in B. burgdorferi-mediated synthesis of uPAR mRNA and protein. Increased uPAR mRNA or protein or both were apparent in PEM from transgenic and control mice, even at a ratio of one Borrelia spirochete per cell. We conclude that signaling for the uPAR response, as mediated by B. burgdorferi, proceeds with CD14 and TLR2 as partial contributors. That part under control of CD14 and TLR2 represents a new link between the host plasminogen activation and innate immunity systems.