The aim of this study was to determine the effect of culture media supplementation with l-carnitine on embryo development and pregnancy rate following cryopreservation. Embryos were produced in vitro using cumulus-oocyte complexes collected by ovum pick-up (OPU) from pregnant, Holstein heifers (n = 24) following superstimulation. Superstimulation was induced 48 h after dominant follicle removal with 2 intramuscular injections of 90 mg of follicle-stimulating hormone (FSH; Folltropin-V) diluted in hyaluronic acid (MAP-5) given 48 h apart. OPU was performed 32 h after the second FSH injection. After fertilization with X-sorted semen, presumptive zygotes (n = 417) were randomly assigned in a crossover design to culture in SOF-BE1 supplemented with 0 or 0.75 mMl-carnitine at 38.5°C in a humidified atmosphere of 5% O₂, 5% CO₂ and 90% N₂. The proportion of oocytes that cleaved was assessed on d 3 after insemination and the proportion of oocytes that developed to the blastocyst stage was determined on d 7. Grade 1 and 2 morula and blastocyst stage (early, blastocyst, expanded and hatched) embryos were harvested on d 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. Lactating Holstein cows were synchronized for timed embryo transfer using the OvSynch-56 protocol (Carvalho et al., 2014). At d 7 after presumptive ovulation, a single embryo (n = 102) was randomly thawed and transferred into cows having a corpus luteum confirmed by ultrasonography. Pregnancy was diagnosed at d 33, 45, and 72 of gestation. Data were analyzed using the GLIMMIX procedure of SAS (P < 0.05). There was no effect of l-carnitine on cleavage rate, blastocyst rate or on the proportion of embryos selected for freezing. Pregnancy rate on d 33, 45 and 72 was not effected by l-carnitine (33.3% ± 0.06 vs. 27.7% ± 0.06, 31.2% ± 0.06 vs. 27.7% ± 0.06, 22.9% ± 0.06 vs. 22.2% ± 0.06 respectively). l-Carnitine also had no effect on pregnancy loss between d 33 and 45 and d 45 and 72 (6.0% ± 0.1 vs. 0.0% and 26.6 ± 0.1 vs. 20.0% ± 0.1, respectively). In conclusion, supplementation of embryo culture media with L-carnitine had no effect on embryo development or pregnancy rate after cryopreservation.