Prostaglandin F (PGF) is commonly injected intramuscularly (IM) in female cattle in synchronization protocols. A novel site for administration of PGF that improves beef quality assurance is the ischiorectal fossa (IRF). The objective of this study was to determine whether administration of PGF in the IRF results in a similar physiological response to an intramuscular injection. Yearling angus-cross heifers (n = 112) were blocked by weight and randomly assigned within blocks to be injected with 5 mL PGF either IM in the neck or in the IRF. Blood samples taken at 0, 8, 16, 24, 36, and 48 h post-injection were analyzed for serum progesterone concentration using a radioimmunoassay. Progesterone concentration curves for each heifer were plotted to determine luteolysis. The median times to luteolysis for neck and IRF injections were 18.1 h and 20.0 h, respectively (p = 0.06). Angus cross commercial beef cows (n = 1471) at least 30 days post-partum were blocked by age and randomly assigned within blocks to be injected with 5 mL PGF either IM in the neck muscle or in IRF as part of a 7-Day CO-Synch + CIDR synchronization protocol. Pregnancy diagnosis was performed via ultrasound at 60 days post insemination. Results were analyzed with Proc Glimmix (SAS). Pregnancy rates for neck and IRF injections were 52.6% and 57.2%, respectively (p = 0.06). In summary, injection of PGF in the IRF for synchronization of estrus and luteolysis did not differ from IM injection. Utilizing the ischiorectal fossa as an injection site for PGF may serve as an alternative that more closely aligns with beef quality assurance.