The myometrium plays a vital role in maintenance of pregnancy. Disruption of myometrial sensitivity to pro-contractile stimuli might lead to preterm labor. Inflammation and/or infection are common precursors to preterm birth, in part by initiating pro-contractile stimuli through toll-like receptor (TLRs) activation. In this study, we investigated the responses specific to inflammatory stimuli for both human primary myometrial cells (HPMCs) and PHM1-41 cells, a human immortalized myometrial cell line. Both these types of cells are commonly used to study labor and pregnancy. Both cell lines were treated with lipopolysaccharide (LPS), peptidoglycan (PGN), or imiquimod (IQ) (ligands for TLRs 2, 4, and 7, respectively). We demonstrate that inflammatory cytokines increase significantly with LPS treatment; however, no change occurs with PGN and IQ, suggesting lack of TLR2- and TLR7-specific signaling in both HPMCs and in the PHM1-41 cell line. Absence of TLR2- and TLR7-specific protein bands on western blots confirmed the lack of these receptors in both HPMCs maintained in long-term culture and PHM1-41 cells. However, TLR2 expression was present in freshly collected matched human myometrial tissue (i.e., the tissues used to create the HPMC cultures), showing loss of TLR2 receptors by HPMCs during the cell culturing process. TLR7 protein expression was lacking both in myometrial tissue and in cultured cells. These results demonstrate the limited applicability and reliability of cellular models to investigate the role of the myometrium during pregnancy and labor.