LPS, a bacterial endotoxin, induces the expression of many genes in macrophages. We report the cloning of a novel 3.3-kb cDNA that is a member of the thymidylate kinase family of genes. This clone, which we have designated TYKi, was obtained by screening a cDNA library prepared from RNA isolated from the murine cell line RAW264.7 after bacterial LPS treatment. TYKi is quite similar to all thymidylate kinases for which there are sequence data. It conserves two very important domains in these kinases, namely, the catalytic domain or P-loop and the nucleotide binding domain. After LPS exposure, the TYKi message appears at 2 h, peaks at 6 h, and declines at 8 h. LPS induction of TYKi is dependent on de novo protein synthesis. Increasing cytosolic cAMP with forskolin attenuates the LPS induction of TYKi. However, treatment with 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) or dibutyryl-cAMP did not affect the LPS induction of TYKi. In contrast, activation of protein kinase C with phorbol ester augmented the LPS response, whereas inhibiting protein kinase C with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) suppressed the LPS response. Removing extracellular Ca2+ with EGTA inhibited LPS induction of TYKi, whereas increasing intracellular calcium with the calcium ionophore A23187 had little effect on the levels of the TYKi transcript. Inhibiting tyrosine kinase with genistein suppressed the induction of TYKi by LPS.