Previously, we showed decreased development of endometriotic lesions in CD44 knockout mice compared to control.1 This suggests that the CD44/hyaluronic acid (HA) system plays a role in the development of the early endometriotic lesion. Here, we assess the in vitro effects of CD44 knockdown (KD) on endometrial epithelial cells (EECs) through attachment to and invasion through a peritoneal mesothelial cell (PMC) monolayer. We also investigate the expression of the receptor for hyaluronic acid mediated motility (RHAMM), another receptor for hyaluronic acid. In vitro studies. Primary EECs were isolated from menstrual endometrial biopsies. EECs were immortalized (iEECs) by telomerase vector, and verified by genotyping. CD44 KD in iEECs was created by CRISPR technique and verified through flow cytometry and western blot. Established in vitro assays were used to assess iEEC attachment to PMCs and invasion through a monolayer of PMCs on a matrigel matrix. Results were analyzed with student t-tests. CD44 KD iEECs demonstrate decreased in vitro attachment to PMCs compared to control (p=0.008). CD44 KD iEECs increased in vitro invasion to PMC compared to control (p= 0.001). In CD44 KD iEECs, RHAMM mRNA expression increases compared to control (p<0.001). Our finding that CD44 KD decreases attachment to PMCs further supports the involvement of CD44/HA in the development of the early endometriotic lesion. The increase in invasiveness of CD44 KD and the compensatory increase in RHAMM suggest that other CD44 ligands are involved in endometrial cell invasion.