We developed a technique of whole cell patch-clamp recordings from white matter oligodendrocytes and astrocytes in 200-250 microm-thick horizontal slices of adult (>2 months, 240-260 g) rat thoracic spinal cord. The viability of the white matter, sectioned in Na(+)-free, low Ca(2+) media, and the function of axons were preserved for >8 h, as demonstrated by the propagation of TTX-sensitive compound action potentials (CAPs) and the sensitivity of their refractory period to K(+) channel blocker 4-aminopyridine (1 microM). Glial cells were visually identified within the slices with a 40 x water immersion objective using infra-red differential interference contrast (IR-DIC) video microscopy, and the details of their morphology were further elucidated after filling the cells with Lucifer Yellow or Alexa 350 fluorescent dyes during whole-cell recording. Using voltage steps and ramps, we revealed pronounced non-linearity of I-V relationships in both oligodendrocytes and astrocytes. Both types of cells expressed TEA-sensitive outward delayed rectifier-type currents activated at positive voltages but showed little, if any, signs of inward rectification at voltages up to -140 mV. At -70 mV holding voltage, bath-applied kainic acid (100 microM) activated inward currents in both types of cells. This novel horizontal slice preparation of adult rat thoracic cord will facilitate the examination of mature glial cell physiology, glial-axonal signaling and the pathophysiology of spinal cord trauma and ischemia.