The recessive mutation 'Heedless' (hdl) was detected in third-generation N-ethyl-N-nitrosourea–mutated mice that showed defective responses to microbial inducers. Macrophages from Heedless homozygotes signaled by the MyD88-dependent pathway in response to rough lipopolysaccharide (LPS) and lipid A, but not in response to smooth LPS. In addition, the Heedless mutation prevented TRAM-TRIF–dependent signaling in response to all LPS chemotypes. Heedless also abolished macrophage responses to vesicular stomatitis virus and substantially inhibited responses to specific ligands for the Toll-like receptor 2 (TLR2)-TLR6 heterodimer. The Heedless phenotype was positionally ascribed to a premature stop codon in Cd14. Our data suggest that the TLR4–MD-2 complex distinguishes LPS chemotypes, but CD14 nullifies this distinction. Thus, the TLR4–MD-2 complex receptor can function in two separate modes: one in which full signaling occurs and one limited to MyD88-dependent signaling.