The production of prostaglandin E2 (PGE2) by human amnion cells in primary monolayer culture was stimulated 2- to 150-fold by mouse epidermal growth factor (mEGF). The effect of mEGF on amnion cell PGE2 production was dependent on the time of treatment and the concentration of mEGF used. The rate of PGE2 production by these cells was maximal between 2 and 4 h of treatment with mEGF; thereafter, the rate of production of PGE2 declined. The stimulation of PGE2 production was maximal at concentrations of mEGF of greater than 5 ng/ml. A number of other growth factors, steroid and protein hormones, and various other test agents were ineffective or only minimally in stimulating PGE2 production by amnion cells. The stimulatory effect of mEGF on PGE2 production in these cells was dependent on the presence of serum in the culture medium; alternatively, mEGF was effective in stimulating PGE2 production by amnion cells in serum-free medium that was supplemented with arachidonic acid bound to albumin. Thus, we conclude that the marked stimulation of PGE2 production by amnion cells treated with mEGF is not due to an action of mEGF to stimulate the release of arachidonic acid from cellular glycerophospholipid storage forms; rather, these data are supportive of the conclusion that mEGF-stimulated PGE2 production is dependent on the presence of nonesterified, i.e., free, arachidonic acid in the medium.