Summary: Our objective was to determine the effect of route of administration of dinoprost tromethamine 7 d after treatment with GnRH on circulating 13,14-dihydro-15-keto-prostaglandin F2α (PGFM) and progesterone (P4) concentrations in lactating dairy cows. Multiparous Holstein cows fitted with indwelling jugular catheters were randomized 7 d after the last GnRH of an Ovsynch protocol (G2, d 0) to receive 25 mg of dinoprost tromethamine either intramuscularly (IM) or subcutaneously (SC). The SC cows had greater circulating PGFM concentrations 15 to 90 min after treatment than the IM cows; however, circulating P4 concentrations during induced luteolysis did not differ at any time based on route of administration.