An inflammatory response is induced in the reproductive tract by the deposition of semen during natural mating. It has been proposed that this response could facilitate establishment and maintenance of pregnancy by modifying the microenvironment of the reproductive tract. The best evidence for this idea comes from mice where females mated with males lacking seminal vesicles experience reduced conception rates, embryos with poor development to the blastocyst stage, low implantation rates, and offspring with altered postnatal phenotype. Here we hypothesized that intrauterine infusion of 0.5 mL of seminal plasma at the time of artificial insemination (AI) in first-service lactating Holstein cows will improve pregnancy rate per insemination. The experiment was conducted at a commercial dairy farm in north-central Florida during the winter (November to February); voluntary waiting period was 82 d. Cows were inseminated [multiparous cows (n = 692), conventional semen; primiparous cows (n = 322), X-sorted semen] using the Double Ovsynch protocol. Cows were randomly assigned to receive intrauterine infusion of either 0.5 mL seminal plasma (prepared as a single pool from 44 bulls) or vehicle (saline) immediately after AI. Pregnancy diagnosis was performed at d 32 after AI. Data were analyzed by the GLIMMIX procedure of SAS. Pregnancy rate per AI was affected by parity (P = 0.0030) and treatment (P = 0.0586) but not by the interaction. Least squares means for multiparous cows were 51.7 + 2.8% for vehicle and 49.0 + 2.6% for seminal plasma. Least squares means for primiparous cows were 45.3 + 3.9% for vehicle and 35.4 + 3.8% for seminal plasma. Results do not support a beneficial effect of seminal plasma on pregnancy rate per AI at first service. Further research to evaluate effects of seminal plasma on pregnancy loss and characteristics of the offspring is warranted. Study supported by Select Sires.