Recent studies suggested that prostaglandin endoperoxide H synthase- and prostaglandin endoperoxide H synthase-2 (PGHS-1 and PGHS-2) utilize different pools of arachidonic acid for synthesizing prostanoids. Using cultured murine NIH3T3 fibroblasts, we investigated the mechanism for the different utilization of arachidonic acid between PGHS-1 and -2. Histofluorescence staining for PGHS activity in intact cells demonstrated that quiescent 3T3 cells expressed only PGHS-1 activity and serum-activated 3T3 cells pretreated with aspirin expressed only PGHS-2 activity. Endogenous arachidonic acid released by calcium ionophore A23187 was not converted by PGHS-1 but exclusively converted by PGHS-2. In the cell free system, the kinetics of PGHS-1 were not so much different from those of PGHS-2. However, in intact cells, arachidonic acid at concentrations lower than 2.5 microM was converted by PGHS-2 alone but not by PGHS-1. Our findings indicated that this small amount of arachidonic acid as released by some stimuli is converted exclusively by PGHS-2. Furthermore, treating the PGHS-2-expressing cells with sodium selenite or ebselen, reducing agents of intracellular peroxides, only decreased PGHS-2 activity. We speculate that only PGHS-2 has been activated by intracellular peroxides and subsequently, it can convert the arachidonic acid released endogenously.