The effects of nicotine on preimplantation embryonic development were investigated in the rat. A high dose of nicotine (7.5 mg nicotine tartrate) was injected s.c. twice daily from the morning of proestrus (Day 0 of pregnancy) until the day of sacrifice. Rats were sacrificed at 600, 1200, 1800 or 2400 h on Days 1–5 of pregnancy and the location and stage of development of the ova were recorded. Nicotine caused a delay of approximately 12 h in ovum cleavage from the 2-cell to the 4-cell stage; each step of embryonic development after the 4-cell stage was thereby delayed. In nicotinetreated rats, ovum entry into the uterus, blastocyst formation, shedding of the zona pellucida and implantation were delayed. A single injection of 0.2 µg estradiol on Day 3 of pregnancy abolished the delay in ovum entry into the uterus. Daily injection of nicotine caused a concentration or “crowding” of implantation sites toward the tubal ends of the uterine horns. An additional injection of estrogen on the afternoon of Day 4 resulted in even greater crowding. Concentrations of progesterone (P), luteinizing hormone (LH) and prolactin (PRL) in peripheral serum during the preimplantation period were lower in nicotine-treated rats than in saline-treated rats. On the other hand, serum concentrations of estrogens and follicle stimulating hormone were higher in nicotine-treated rats as compared to those in saline-treated rats. These results suggest that delayed implantation of ova in nicotine-treated rats is due not to a decreased secretion of estrogen, but rather to a delay in the increase of P secretion which is necessary to prepare the uterus for implanting blastocysts. This disturbance in P secretion appears to be the result, at least in part, of the hormonal imbalance of the hypothalamo-pituitary axis caused by the nicotine treatment.