The purpose of this study was to test the hypothesis that specific macrophage-secreted cytokines cause gene expression changes in endometrial stromal cells that reproduce the effects of macrophages in the development of endometriosis. Telomerase-immortalized human endometrial stromal cells (T-HESC) were treated with tumor necrosis factor α (TNFα, 5 ng/ml) and interleukin 1β (IL1β, 1 ng/ml). Differential expression of 249 genes was identified by DNA microarray. Ontologies such as peptidases, cell adhesion, cell death/cell cycle, growth factors, cytoskeletal organization, defense/immune system, signal transduction, and transcriptional regulation which are related to the development of endometriosis were represented by these genes. The up-regulation of interleukin 8 (IL8), interleukin 6 (IL6), IL1β and matrix metalloproteinase 3 (MMP3) in response to TNFα ± ILIβ in T-HESC cells was confirmed by real time RT-PCR. TNFα ± ILIβ did not affect the migration or invasion of T-HESC cells. This study reinforces our previous investigations on communication between cells of the immune system and endometrial stromal cells and their potential role in the development of endometriosis.