The present study was undertaken to establish whether mouse uterine epithelial cells produce CCL20/macrophage inflammatory protein 3α (CCL20/MIP-3α) and to determine whether secretion is under hormonal control and influenced by pathogen-associated molecular patterns (PAMPs). In the absence of PAMPs, polarized uterine epithelial cells grown to confluence on cell culture inserts constitutively secreted CCL20/MIP-3α with preferential accumulation into the apical compartment. When epithelial cells were treated with the Toll-like receptor (TLR) agonists Pam3Cys (TLR2/1), peptidoglycan (TLR2/6) or lipopolysaccharide (LPS; TLR4), CCL20/MIP-3α increased rapidly (4 hr) in both apical and basolateral secretions. Time–course studies indicated that responses to PAMPs added to the apical surface persisted for 12–72 hr. Stimulation with loxoribin (TLR7) and DNA CpG motif (TLR9) increased basolateral but not apical secretion of CCL20/MIP-3α. In contrast, the viral agonist Poly(I:C) (TLR3) had no effect on either apical or basolateral secretion. In other studies, we found that oestradiol added to the culture media decreased the constitutive release of CCL20/MIP-3α. Moreover, when added to the culture media along with LPS, oestradiol inhibited LPS-induced increases in CCL20/MIP-3α secretion into both the apical and basolateral compartments. In summary, these results indicate that CCL20/MIP-3α is produced in response to PAMPs. Since CCL20/MIP-3α is chemotactic for immature dendritic cells, B cells and memory T cells and has antimicrobial properties, these studies suggest that CCL20/MIP-3α production by epithelial cells, an important part of the innate immune defence in the female reproductive tract, is under hormonal control and is responsive to microbial challenge.