To investigate the possible role of prostaglandins in implantation in rats, indomethacin, an inhibitor of prostaglandin synthesis, was given to rats at 0800 and 1300 h on Day 5 of pregnancy. The uterine dye site reaction, indicative of the initiation of implantation, was present on the evening of Day 5 in 10/10 and 1/10 control and indomethacin-treated rats, respectively. Indomethacin treatment on Day 5 did not terminate pregnancy; on Day 8 there was no significant difference between control and treated rats in the proportions pregnant or in the numbers of implantation sites. However, implantation sites weighed significantly less and the length of pregnancy was slightly longer in treated rats, suggesting that indomethacin treatment had delayed implantation. Implantation in treated rats was not initiated without an increase in endometrial capillary permeability; rats treated with indomethacin on Day 5 and killed on the morning of Day 6 had uterine dye sites which were smaller and less evenly spaced than those of control animals killed at the same time. For rats ovariectomized on Day 3, treated with progesterone and given estradiol-17β to induce implantation, the uterine dye site reaction was present in 9/9 and 1/9 control and indomethacin-treated rats, respectively, suggesting that the indomethacin effect was not mediated by inhibition of ovarian steroidogenesis. In addition, serum progesterone levels on Day 5 were nor influenced by indomethacin treatment. The concentrations of prostaglandins of both the E and F series were significantly greater in the dye site areas than elsewhere in the uterus on the evening of Day 5. The results of these experiments suggest that prostaglandins are involved at the uterine level in the initiation of implantation in the rat.