It is study was designed to characterize endotoxin-induced prostaglandin production in primary cultured rat vascular smooth muscle cells (VSMC). The time course for prostag)andim synthesis in lipopolysaccharide (LPS)-stimulated VSMC showed that the maximum production was reached in 12 hours. LPS induced prostaglandin H2 synthase (PGHS) activity in VSMC and the time course profile in the changes of PGHS activity paralleled that of total prostaglandin production. Differential treatment showed that 4 hours' exposure to LPS was enough for the maximum effect on the prostaglandin production and this effect was completely inhibited by the co-treatment of actinomycin D, a transcription inhibitor. These results suggest that LPS effect might be determined within 4 hours. Actinomycin D increased PGHS activity without affecting prostaglandin production if added 4 hours after LPS treatment. On the other hand, cycloheximide, a translation inhibitor, augmented LPS-induced prostaglandin production if treated during first four hours, but it inhibited LPS-induced PGHS activity regardless of treatment schedule. These results suggeat the existence of multiple regulating mechanisms in the LPS-induced prostaglandin synthesis.