Otitis media is one of the most common and intractable ear diseases, and is the major cause of hearing loss, especially in children. Multiple factors affect the onset or development of otitis media. Prostaglandin D-2 is the major prostanoid involved in infection and allergy. However, the role of prostaglandin D-2 and prostaglandin D2 receptors on the pathogenesis of otitis media remains to be determined. Recent studies show that D prostanoid receptor (DP) and chemoattractant receptor-homologous molecule expressed on T helper type 2 (Th2) cells (CRTH2) are major prostaglandin D-2 receptors. In this study, homozygous DP single gene-deficient (DP-/-) mice, CRTH2 single gene-deficient (CRTH2(-/-)) mice and DP/CRTH2 double gene-deficient (DP-/- CRTH2(-/-)) mice were used to investigate the role of prostaglandin D-2 and its receptors in otitis media. We demonstrate that prostaglandin D-2 is induced by lipopolysaccharide (LPS), a major component of Gram-negative bacteria, and that transtympanic injection of prostaglandin D-2 up-regulates macrophage inflammatory protein 2 (MIP-2), interleukin (IL)-1 beta and IL-6 in the middle ear. We also show that middle ear inflammatory reactions, including infiltration of inflammatory cells and expression of MIP-2, IL-1 beta and IL-6 induced by LPS, are reduced significantly in DP-/- mice and DP-/- CRTH2(-/-) mice. CRTH2(-/-) mice display inflammatory reactions similar to wild-type mice. These findings indicate that prostaglandin D-2 may play significant roles in LPS-induced experimental otitis media via DP.