The aim of the present work was to study the topography and structure of 28-kDa calbindin (CB)-containing interneurons in the ventral horn and intermediate zone of the gray matter at levels TII and LIV of the spinal cord (SC). Experiments were performed in adult female Wistar rats (n = 4) using immunohistochemical and morphometric methods. Cryostat transverse sections of the SC of thickness 14 μm were studied. The intermediate zone of the SC was found to contain two subpopulations of CB-immunoreactive (IR) interneurons in lamina VII in both segments: sympathetic preganglionic neurons and “partition” cells. The ventral horn also contained two subpopulations of CB-IR interneurons: Renshaw cells in lamina IX in TII and lamina VII in LIV and large interneurons in lamina VIII in TII and lamina VII in LIV. The only differences between segments were in the larger numbers of Renshaw cells and partition cells in LIV. The mean cross-sectional areas of CB-IR Renshaw cells and autonomic preganglionic neurons were greater in TII, while large interneurons of the ventral horn and partition cells were larger in LIV.