Mammals mount a rapid inflammatory response to gram-negative bacteria by recognizing lipopolysaccharide (LPS, endotoxin). LPS binds to CD14, and the resulting LPS-CD14 complex induces synthesis of cytokines and up-regulation of adhesion molecules in a variety of cell types. Gram-positive bacteria provoke a very similar inflammatory response, but the molecules that provoke innate responses to these bacteria have not been defined. Here we show that protein-free, phenol extracts of Staphylococcus aureus contain a minor component that stimulates adhesion of neutrophils and cytokine production in monocytes and in the astrocytoma cell line, U373. Responses to this component do not absolutely require CD14, but addition of soluble CD14 enhances sensitivity of U373 cells by up to 100-fold, and blocking CD14 on monocytes decreases sensitivity nearly 1,000-fold. Deletion of residues 57-64 of CD14, which are required for responses to LPS, also eliminates CD14-dependent responses to S. aureus molecules. The stimulatory component of S. aureus binds CD14 and blocks binding of radioactive LPS. Unlike LPS, the activity of S. aureus molecules was neither enhanced by LPS binding protein nor inhibited by bactericidal/permeability increasing protein. The active factor in extracts of S. aureus is also structurally and functionally distinct from the abundant species known as lipoteichoic acid (LTA). Cell-stimulating activity fractionates differently from LTA on a reverse-phase column, pure LTA fails to stimulate cells, and LTA antagonizes the action of LPS in assays of IL-6 production. These studies suggest that mammals may use CD14 in innate responses to both gram-negative and gram-positive bacteria, and that gram-positive bacteria may contain an apparently unique, CD14-binding species that initiates cellular responses.