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In ruminants, uterine pulses of prostaglandin (PG) F2αcharacterize luteolysis, while increased PGE2/PGE1distinguish early pregnancy. This study evaluated intrauterine (IU) infusions of PGF2αand PGE1pulses 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), PGF2α(0.25 mg PGF2α), or PGE1+ PGF2α. A luteal biopsy was collected at 30 min after third infusion for determination of gene expression by RNA-Seq. As expected, IU pulses of PGF2αdecreased (P< 0.01) P4 luteal volume. However, there were no differences in circulating P4 or luteal volume between saline, PGE1, and PGE1+ PGF2α, indicating inhibition of PGF2α-induced luteolysis by IU pulses of PGE1. After third pulse of PGF2α, 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 PGE1or PGE1+ PGF2α, there were no significant changes in luteal gene expression (FDR > 0.10) compared to saline cows. Increased circulating concentrations of the metabolite of PGF2α(PGFM; after PGF2αand PGE1+ PGF2α) and the metabolite PGE (PGEM; after PGE1and PGE1+ PGF2α) demonstrated that PGF2αand PGE1are entering bloodstream after IU infusions. Thus, IU pulses of PGF2αand PGE1allow 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 PGF2αtransport to the CL or PGF2αaction by PGE1resulting in complete inhibition of transcriptomic changes following IU PGF2αpulses.Treatment with PGF2αinduced dramatic changes in expression of 955 genes, based on RNA-Seq after the third PGF2αpulse (FDR < 0.01), whereas simultaneous treatment with pulses of PGE1blocked luteolysis and gene expression induced by PGF2α.