LPS induces in bone marrow macrophages the transient expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). Because MKP-1 plays a crucial role in the attenuation of different MAPK cascades, we were interested in the characterization of the signaling mechanisms involved in the control of MKP-1 expression in LPS-stimulated macrophages. The induction of MKP-1 was blocked by genistein, a tyrosine kinase inhibitor, and by two different protein kinase C (PKC) inhibitors (GF109203X and calphostin C). We had previously shown that bone marrow macrophages express the isoforms PKCβI, ε, and ζ. Of all these, only PKCβI and ε are inhibited by GF109203X. The following arguments suggest that PKCε is required selectively for the induction of MKP-1 by LPS. First, in macrophages exposed to prolonged treatment with PMA, MKP-1 induction by LPS correlates with the levels of expression of PKCε but not with that of PKCβI. Second, Gö6976, an inhibitor selective for conventional PKCs, including PKCβI, does not alter MKP-1 induction by LPS. Last, antisense oligonucleotides that block the expression of PKCε, but not those selective for PKCβI or PKCζ, inhibit MKP-1 induction and lead to an increase of extracellular-signal regulated kinase activity during the macrophage response to LPS. Finally, in macrophages stimulated with LPS we observed significant activation of PKCε. In conclusion, our results demonstrate an important role for PKCε in the induction of MKP-1 and the subsequent negative control of MAPK activity in macrophages.