Purified components from bacteria selectively activate Toll-like receptors (TLR), leading to shared and unique responses in innate immune cells. Whole bacteria contain agonists for multiple TLR and induce a common macrophage activation program of transcription. It is not known, however, whether the stimulation of specific TLR by whole bacteria results in differential activation of the innate immune system. We evaluated gene expression data from human macrophages and found a unique gene expression profile induced by Gram-negative bacteria. In contrast, Gram-positive bacteria evoked few specific alterations in gene expression. LPS, a TLR4-specific ligand, was sufficient to elicit the distinct expression profile observed with Gram-negative bacteria. TLR4 activation regulated gene expression by both an IFN-dependent and an IFN-independent mechanism, illustrated by I-TAC and IL-12 p70, respectively. IL-12 p70 was produced by cells in whole blood exposed to Gram-negative bacteria, demonstrating faithful reproduction of the macrophage response in mixed populations of cells and identifying a potential diagnostic marker of infection. Our results show that the macrophage response to bacteria is dominated by the accumulated input from multiple TLR. For macrophages exposed to Gram-negative bacteria, gene expression changes encompass those induced by Gram-positive bacteria plus a distinct TLR4 response. This distinct TLR4 response may provide the basis to diagnose clinical Gram-negative infections.