TLRs are a major group of pattern recognition receptors that are crucial in initiating innate immune responses and are capable of recognizing Plasmodium ligands. We have investigated TLR responses during acute experimental P. falciparum (P.f.) infection in 15 malaria-naive volunteers. TLR-4 responses in whole blood ex vivo stimulations were characterized by significantly (p < 0.01) up-regulated proinflammatory cytokine production during infection compared with baseline, whereas TLR-2/TLR-1 responses demonstrated increases in both proinflammatory and anti-inflammatory cytokine production. Responses through other TLRs were less obviously modified by malaria infection. The degree to which proinflammatory TLR responses were boosted early in infection was partially prognostic of clinical inflammatory parameters during the subsequent clinical course. Although simultaneous costimulation of human PBMC with P.f. lysate and specific TLR stimuli in vitro did not induce synergistic effects on cytokine synthesis, PBMC started to respond to subsequent TLR-4 and TLR-2 stimulation with significantly (p < 0.05) increased TNF-alpha and reduced IL-10 production following increasing periods of preincubation with P.f. Ag. In contrast, preincubation with preparations derived from other parasitic, bacterial, and fungal pathogens strongly suppressed subsequent TLR responses. Taken together, P.f. primes human TLR responses toward a more proinflammatory cytokine profile both in vitro and in vivo, a characteristic exceptional among microorganisms.