There exists no distinct explanation for the prepartal prostaglandin F2α (PGF2α) increase mechanism in dogs. While the PGF2α-synthase (PGFS) mRNA expression and localization profiles have been previously investigated in canine utero/placental compartment, the availability and biochemical activity of the PGFS protein remains unknown. Consequently, in order to provide a basis for better understanding of mechanisms controlling the provision of prostaglandins, canine-specific PGFS- and 15-Prostaglandin Dehydrogenase (15PGDH)- antibodies were generated and used in the present study. The spatio-temporal expression and biochemical activities of recombinant canine PGFS and 15PGDH were investigated throughout pregnancy. From the strong expression of PGFS observed in the utero/placental compartment during post-implantation and mid-gestation, which reflects the mRNA-levels observed previously, and its localization in the placenta fetalis, the possible role of PGF2α in the trophoblast invasion during canine placentation is suggested. Similar localization pattern, together with the increased expression from pre-implantation until mid-gestation, were observed for 15PGDH. Cumulatively, the presented results imply a possible functional interplay between PGFS and 15PGDH controlling the placental development and prepartum PGF2α output in the dog. 15PGDH appears to be a potential “gate-keeper” in the local provision of prostaglandins from the pregnant canine uterus.