Midazolam, a benzodiazepine receptor agonist, when injected intrathecally either enhances or decreases antinociception produced by intrathecal administration of morphine in rats. Furthermore, midazolam inhibits binding of several opioid ligands to spinal opioid receptors in vitro [Rattan et al, Anesth Analg 1991;73:124–131]. This study was designed to investigate the effect of midazolam on binding of µ-, δ- and ĸ-ligands to rat spinal opioid receptors in the presence of sodium ions which differentially modulate binding of opioid agonists and antagonists. Sodium ions (50-1,000 mmol/l) selectively increased the specific binding of [3H]naloxone but decreased binding of opioid agonists such as [3H]DAGO (Tyr-D-Ala-Gly-Methyl-Phe-Gly-ol-enkephalin) to µ-receptors, [3H]DSTLE (Tyr-D-Ser-Gly-Phe-Leu-Thr-enkephalin) to δ-receptors and [3H]EKC (ethylketocyclazocine) to ĸ-receptors in rat spinal cord in vitro. Midazolam (1-100 µmol/l) inhibited the binding of [3H]naloxone, [3H]DAGO, [3H]DSTLE and [3H]EKC. Sodium ions (100 mmol/l) antagonized the inhibition of binding of [3H]naloxone and [3H]DSTLE by midazolam by increasing IC50 values for midazolam. However, sodium ions potentiated the inhibition of binding of [3H]DAGO by midazolam by decreasing IC50 value for midazolam and had a mixed effect on binding of [3H]EKC in the presence of midazolam. Scatchard analysis performed in the presence of sodium ions and/or midazolam confirmed the specific effects of sodium ions as well as midazolam on the Bmax and Kd of µ-, δ-, and K-receptors. These results suggest for the first time that sodium ions play an important role in the modulation of spinal opioid receptors by benzodiazepines. Sodium ions potentiate the inhibition of DAGO binding but antagonize the inhibition of naloxone and DSTLE binding by midazolam in rat s