The contribution of hepatocytes to liver prostaglandin (PG) synthesis Is not clear. We compared prostaglandin synthesis in homogenates of whole liver, freshly isolated hepatocytes, and mixed non-parenchymal cells from the same rat livers, and optimized the assay. Whole liver homogenates made 27.2 +/- 7.1 mg PGE 2 /mg protein/5 min (+/- SEM, n = 4 livers). Hepatocyte homogenates made 39 +/- 9% as much PGE 2 /mg protein as did the matched whole livers. Non-parenchymal cell homogenates made slightly more PGE 2 than whole liver, but much more PGD 2 . Subsequent studies showed that fresh hepatocyte suspensions contain significant contamination with non-parenchymal cells. Homogenates from ricin-purified hepatocyte monolayers made at least half as much PGE 2 as did conventional monolayers. However, taking cellular purity into account, hepatocytes must contain much less than a third of liver cyclooxygenase activity.