In ruminants, uterine pulses of prostaglandin (PG) F 2α characterize luteolysis, while increased PGE 2 /PGE 1 distinguish early pregnancy. This study evaluated intrauterine (IU) infusions of PGF 2α and PGE 1 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, PGE 1 (2 mg PGE 1), PGF 2α (0.25 mg PGF 2α), or PGE 1 + 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, PGE 1, and PGE 1 + PGF 2α, indicating inhibition of PGF 2α -induced luteolysis by IU pulses of PGE 1. 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 PGE 1 or PGE 1 + 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 PGE 1 + PGF 2α) and the metabolite PGE (PGEM; after PGE 1 and PGE 1 + PGF 2α) demonstrated that PGF 2α and PGE 1 are entering bloodstream after IU infusions. Thus, IU pulses of PGF 2α and PGE 1 allow determination of changes in luteal gene expression that could be relevant to understanding luteolysis and pregnancy. Unexpectedly, by third pulse of PGE 1, there is complete blockade of either PGF 2α transport to the CL or PGF 2α action by PGE 1 resulting in complete inhibition of transcriptomic changes following IU PGF 2α pulses. © The Author(s) 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail:.