Objective To determine the role of peritoneal mesothelial cells (PMCs) in the process of endometrial invasion into the peritoneum and to evaluate gene expression after endometrial-PMC co-culture. Design In vitro study. Setting University laboratory. Patient(s) Reproductive-age women without endometriosis. Intervention(s) None. Main Outcome Measure(s) The rate of endometrial invasion through modeled peritoneum in the presence and absence of PMCs was evaluated. The influence of endometrial-PMC attachment on the expression of target genes, implicated in the pathogenesis of endometriosis, was examined by using reverse transcription polymerase chain reaction. Result(s) Endometrial stromal cell (ESC) invasion through invasion chambers coated with Matrigel (MTGL) and with growth factor–reduced Matrigel (GFR-MTGL) was increased 10-fold when a PMC monolayer was present. Endometrial epithelioid cell (EM42) invasion increased greater than threefold through the MTGL and GFR-MTGL–coated membranes when a PMC monolayer was present. Endometrial stromal cell, EM42, and PMC transcription of extracellular signal-related kinase, colony stimulating factor-1, c-fms, and c-Met was increased after endometrial-PMC attachment. Similar changes were not seen when endometrial cells were exposed to PMC-conditioned media and when PMCs were exposed to endometrial cell conditioned media. Conclusion(s) Peritoneal mesothelial cells increased invasion of ESCs and EM42s through modeled peritoneum. Endometrial-PMC co-culture led to alterations in gene transcription by endometrial cells and PMCs. This study suggests that PMCs contribute to the process of endometrial invasion into the peritoneum.