Changes in patterns of gene induction by myeloid lineage cells following multiple exposures to endotoxin (lipopolysaccharide; LPS) is a feature of LPS tolerance. To further understand the mechanism of this phenomenon we describe studies using stably transfected Chinese hamster ovary cell lines that express human CD14 (CHO-hCD14). Using NF-kappa B activation as a measure of LPS-induced cell activation we show that a single treatment with LPS renders CHO-hCD14 cells tolerant to subsequent challenge with LPS, but not with other stimuli such as tumor necrosis factor. Tolerance may result from the induction of gene(s) that control LPS-induced signaling pathways and here we suggest that such genes may be found in the group of immediate, early response genes characterized by the protein phosphatase 3CH134. The CHO-hCD14 cell lines provide a novel model system to further explore the mechanism of endotoxin tolerance.