The objectives of this study were to optimize the isolation of luteal endothelial cells (LEC) and examine their functional interactions with autologous T lymphocytes. Analysis by flow cytometry showed that the purity of LEC isolated by filtration was nearly 90% as indicated by (BS)-1 lectin binding. LEC expressed mRNA for progesterone receptor (PGR), prostaglandin receptors (PTGFR, PTGER2 and 4, and PTGIR), tumor necrosis factor receptors (TNFRSF1A&B) and interleukin (IL) 1B receptors (IL1R1&2). LEC were pretreated with either vehicle, progesterone (P; 0-20 µM), prostaglandin (PG) E or PGF (0-0.2 µM), and further treated with or without TNF and IL1B (50 ng/mL each). LEC were then incubated with autologous T lymphocytes in an adhesion assay. Fewer lymphocytes adhered to LEC after exposure to high compared to low P concentrations (cubic response; < 0.05). In contrast, 0.2 µM PGE and PGF each increased T lymphocyte adhesion in the absence of cytokines ( < 0.05). LEC induced IL2 receptor alpha (CD25) expression and proliferation of T lymphocytes. In conclusion, filtration is an effective way of isolating large numbers of viable LEC. It is proposed that PGs and P modulate the ability of endothelial cells to bind T lymphocytes, potentially regulating extravasation, and that LEC activate T lymphocytes migrating into or resident in the CL.