In ruminants, uterine pulses of prostaglandin (PG) F_2α characterize luteolysis, while increased PGE₂/PGE₁ distinguish early pregnancy. This study evaluated intrauterine (IU) infusions of PGF_2α and PGE₁ pulses on corpus luteum (CL) function and gene expression. Cows on day 10 of estrous cycle received 4 IU infusions (every 6 h; n = 5/treatment) of saline, PGE₁ (2 mg PGE₁), PGF_2α (0.25 mg PGF_2α), or PGE₁ + PGF_2α. A luteal biopsy was collected at 30 min after third infusion for determination of gene expression by RNA-Seq. As expected, IU pulses of PGF_2α decreased (P < 0.01) P4 luteal volume. However, there were no differences in circulating P4 or luteal volume between saline, PGE₁, and PGE₁ + PGF_2α, indicating inhibition of PGF_2α-induced luteolysis by IU pulses of PGE₁. After third pulse of PGF_2α, luteal expression of 955 genes were altered (false discovery rate [FDR] < 0.01), representing both typical and novel luteolytic transcriptomic changes. Surprisingly, after third pulse of PGE₁ or PGE₁ + PGF_2α, there were no significant changes in luteal gene expression (FDR > 0.10) compared to saline cows. Increased circulating concentrations of the metabolite of PGF_2α (PGFM; after PGF_2α and PGE₁ + PGF_2α) and the metabolite PGE (PGEM; after PGE₁ and PGE₁ + PGF_2α) demonstrated that PGF_2α and PGE₁ are entering bloodstream after IU infusions. Thus, IU pulses of PGF_2α and PGE₁ allow determination of changes in luteal gene expression that could be relevant to understanding luteolysis and pregnancy. Unexpectedly, by third pulse of PGE₁, there is complete blockade of either PGF_2α transport to the CL or PGF_2α action by PGE₁ resulting in complete inhibition of transcriptomic changes following IU PGF_2α pulses.