Two unilateral injections of pH 4.0 saline into the gastrocnemius muscle result in a bilateral decrease in mechanical withdrawal threshold after the second injection. This decrease is significant by 4 h and lasts through 1 week. The purpose of this study was to characterize the involvement of both N-methyl- d-aspartate (NMDA) and non-NMDA glutamate receptors in the spinal cord dorsal horn in the development and maintenance of mechanical hyperalgesia from repeated intramuscular injections of acidic saline. 2-amino-5-phosphonovaleric acid (AP5) (2–20 nmol, 10 μl, pH 7) or 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo[f]quinoxaline-7-sulfonamide (NBQX) (1–10 nmol, 10 μl, pH 8–9) was administered intrathecally to the lumbar spinal cord to block NMDA and non-NMDA ionotropic glutamate receptors in the dorsal horn, respectively. Drugs were administered at one of three different time points: (1) prior to the first intramuscular injection of pH 4.0 saline on Day 0, (2) prior to the second intramuscular injection of pH 4.0 saline on Day 5, and (3) 1 week after the second injection. Mechanical withdrawal thresholds were measured with von Frey filaments before, 4 h, and 24 h after injection 1 and before, 4 h, 24 h, and 1 week after injection 2. AP5 had no effect on mechanical withdrawal thresholds when administered prior to the first intramuscular injection of pH 4.0 saline. When AP5 was administered before the second intramuscular injection, the bilateral decrease in mechanical withdrawal thresholds was delayed for up to 24 h. Intrathecal administration of AP5 1 week after the second intramuscular injection of pH 4.0 saline produced a bilateral increase in mechanical withdrawal thresholds. Blockade of non-NMDA glutamate receptors in the spinal cord dorsal horn prior to either the first or second intramuscular injection of pH 4.0 saline had no effect on the development of mechanical hyperalgesia. However, spinal injection of NBQX 1 week after the second intramuscular injection of pH 4.0 saline resulted in an increase in mechanical withdrawal thresholds when compared to vehicle controls. These data suggest that both NMDA and non-NMDA glutamate receptors are involved in the maintenance of chronic, muscle-induced hyperalgesia.