The superficial dorsal horn of the spinal cord, particularly lamina I, plays a key role in the integration and relay of pain related sensory input. To study the physiology of lamina I neurons in slices, a clear delineation of this layer can be greatly advantageous. Yet, it has remained difficult to distinguish this layer in live tissue in conventional transverse spinal slices because of its very narrow thickness at the edge of the dorsal horn. We describe here the criteria we used to delineate lamina I in live tissue using gradient contrast videomicroscopy in 400 microm-thick parasagittal spinal cord slices from adult rats (30-60-day-old). Because of the longitudinal orientation of the neurons in this layer, the resulting distinctive reticulated appearance of lamina I made it possible to readily distinguish it from lamina II. The usefulness of this distinguishing parameter is demonstrated by our ability to contrast synaptic properties of neurons in lamina I from those in lamina II. Complete morphological identification of lamina I neurons however also requires visualization of the cell in the horizontal plane. To maintain compatibility with the parasagittal slice, we used 3D reconstructions from confocal images of the recorded neurons. Rotation of the neuron in space allowed for its morphological characterization in all three planes (horizontal, parasagittal, and transverse). This approach therefore presents optimal conditions for systematic electrophysiological recording from visually identified lamina I neurons.