The enteric nervous system plays an integral role in the gastrointestinal tract. Within this intricate network, enteric glia are crucial in the maintenance of normal bowel function, yet their signaling mechanisms are poorly understood. Enteric glia, and not enteric neurons, selectively responded to lysophosphatidic acid (LPA), a product of phosphatidylcholine metabolism, with dose-dependent calcium (Ca²⁺) signaling over a range from 100 pM to 10 μM. The elicited calcium transients involved both the mobilization of intracellular Ca²⁺ stores and the influx of extracellular Ca²⁺ as LPA signals were obliterated following the depletion of intracellular Ca²⁺ and attenuated by the removal of Ca²⁺ from the perfusion buffer. Pretreatment with pertussis toxin (100 ng/ml) reduced the magnitude of LPA Ca²⁺ transients (95±20 nM vs 168±17 nM for controls). Repetitive exposure yielded diminished responsiveness, with a 25% reduction in [Ca²⁺]_i between first and second exposures. Inhibition of the inositol 1,4,5-trisphosphate (IP₃) receptor with 200 μM 2-aminoethoxydiphenylborate (2APB) abolished LPA signals. RT-PCR analysis demonstrated the presence of two LPA-coupled endothelial differentiation gene (EDG) receptor mRNAs (EDG-2 and EDG-7) in myenteric plexus primary cultures. EDG-2 expression in glial cells of the ENS was confirmed immunocytochemically.