The role of estrogen modulation of N-methyl-D-aspartate (NMDA) receptor signaling of colorectal pain in the spinal cord was studied in ovariectomized (OVx) and OVx with estradiol replacement (E2) rats. The magnitude of the visceromotor response (vmr) to noxious colorectal distention (CRD) is significantly greater in E2 rats compared to OVx rats. Estrogen receptor alpha and the NR1 subunit of the NMDA receptor are co-expressed in dorsal horn neurons, supporting a direct action of estrogen on NMDA receptor function. Intrathecal administration of the NMDA receptor antagonist D(-)-2-amino-5-phosphonopetanoic acid (APV) dose-dependently attenuated the vmr, but the attenuation was greater in OVx compared to E2 rats. Western blots revealed the total amount of NR1 protein in the lumbosacral spinal cord was significantly greater in E2 rats compared to OVx rats. Phosphorylation of Ser897, a PKA phosphorylation site on the NR1 subunit was significantly increased in E2, but not OVx rats in response to CRD. Phosphorylation of NR1 Ser896 was not changed in either group. Activation (phosphorylation) of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) paralleled phosphorylation pNR1 Ser897. Intrathecal administration of the PKA inhibitors H89 and Rp-8-Br-cAMP significantly attenuated the vmr, decreased NR1 and ERK1/2 phosphorylation and increased the potency of APV to attenuate the vmr compared to vehicle-treated E2 rats. These data suggest that estrogen increases spinal processing of visceral pain by increasing the number of NMDA receptors and increasing site specific receptor phosphorylation on the NMDA NR1 subunit contributing to an increase in NMDA receptor activity. Supported by NS 37424 and AR 49555.