Stimulated production of reactive oxygen species (ROS) by plasma membrane-associated nicotinamide adenine dinucleotide phosphate oxidases (Nox) in non-phagocytic cells regulates a number of biological processes including growth, vessel tone, and oxygen sensing. The purpose of this study was to investigate H(2)O(2)-stimulated ROS production in primary adult cardiac fibroblasts (CF). Results demonstrate that CF express an H(2)O(2)-inducible oxidant generating system that is inhibitable by diphenylene iodonium (DPI) and sensitive to antioxidants. In addition to H(2)O(2), generation of ROS was stimulated potently by 1-oleoyl-2-acetyl-sn-glycerol (OAG) and arachidonic acid (AA) in a protein kinase C-independent manner. Pretreatment with arachidonyl trifluoromethyl ketone was nearly as effective as DPI at reducing H(2)O(2)- and OAG-stimulated oxidant generation indicating a central role for phospholipase A(2) (PLA(2)) in this signaling pathway. Co-stimulation with H(2)O(2) and OAG did not increase ROS generation as compared to OAG alone suggesting both agonists signal through a shared, rate-limited enzymatic pathway involving PLA(2). Co-stimulation with H(2)O(2) and AA had additive effects indicating these two agonists stimulate oxidant production through a parallel activation pathway. Reverse transcriptase-coupled polymerase chain reaction and Western blotting demonstrate primary cardiac fibroblasts express transcripts and protein for Nox4, p22, p47, and p67 phox. Transfections with Nox4 small inhibitory ribonucleic acid oligonucleotides or p22 phox antisense oligonucleotides significantly downregulated stimulated Nox activity. Inhibitors of nitric oxide synthases were without effect. We conclude adult CF express Nox4/p22 phox-containing oxidant generating complex activated by H(2)O(2), OAG, and AA through a pathway that requires activation of PLA(2).