We examined herein the functional linkage of enzymes regulating the initial, intermediate, and terminal steps of PG biosynthesis to provide PGs in rat peritoneal macrophages stimulated with LPS and/or A23187. Quiescent cells stimulated with A23187 produced thromboxane B 2 (TXB 2) in marked preference to PGE 2 within 30 to 60 min (constitutive immediate response), which was mediated by preexisting cytosolic phospholipase A 2 (cPLA 2), cyclooxygenase-1 (COX-1), and TX synthase. Cells treated with LPS predominantly produced PGE 2 during culture for 3 to 24 h (delayed response), where cPLA 2 and secretory PLA 2 functioned cooperatively with inducible COX-2, which was, in turn, coupled with inducible PGE 2 synthase. Cells primed for 12 h with LPS and stimulated for 30 min with A23187 produced PGE 2 in marked preference to TXB 2 (induced immediate response), in which three inducible enzymes, cPLA 2, COX-2, and PGE 2 synthase, were functionally linked. Preferred coupling of the two inducible enzymes, COX-2 and PGE 2 synthase, was further confirmed by the ability of LPS-treated cells to convert exogenous arachidonic acid to PGE 2 optimally at a time when both enzymes were simultaneously induced. These results suggest that distinct PG biosynthetic enzymes display segregated functional coupling following different transmembrane stimulation events even when enzymes that catalyze similar reactions in vitro coexist in the same cells.