The anti-inflammatory drug licofelone [=ML3000; 2-[6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl] acetic acid], currently undergoing phase III trials for osteoarthritis, inhibits the prostaglandin (PG) and leukotriene biosynthetic pathway. Licofelone was reported to suppress the formation of PGE(2) in various cell-based test systems, but the underlying molecular mechanisms are not entirely clear. Here, we examined the direct interference of licofelone with enzymes participating in PGE(2) biosynthesis, that is, cyclooxygenase (COX)-1 and COX-2 as well as microsomal PGE(2) synthase (mPGES)-1. Licofelone concentration-dependently inhibited isolated COX-1 (IC(50) = 0.8 microM), whereas isolated COX-2 was less affected (IC(50) > 30 microM). However, licofelone efficiently blocked the conversion of PGH(2) to PGE(2) mediated by mPGES-1 (IC(50) = 6 microM) derived from microsomes of interleukin-1beta-treated A549 cells, being about equipotent to 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid (MK-886), a well recognized mPGES-1 inhibitor. In intact interleukin-1beta-treated A549 cells, licofelone potently (IC(50) < 1 microM) blocked formation of PGE(2) in response to calcimycin (A23187) plus exogenous arachidonic acid, but the concomitant generation of 6-keto PGF(1alpha), used as a biomarker for COX-2 activity, was not inhibited. We conclude that licofelone suppresses inflammatory PGE(2) formation preferentially by inhibiting mPGES-1 at concentrations that do not affect COX-2, implying an attractive and thus far unique molecular pharmacological dynamics as inhibitor of COX-1, the 5-lipoxygenase pathway, and of mPGES-1.