Double immunofluorescence was utilized to determine whether Renshaw cells contain calbindin D28k immunoreactivity. Renshaw cells were identified by their characteristic expression patterns of gephyrin immunoreactivity in sections of rat and cat lumbar spinal cord. In the rat, all neurons classified as Renshaw cells (n = 487) also contained calbindin D28k-immunoreactivity, and all calbindin D28k-immunoreactive cells located in the ventral-most region of lamina VII expressed the characteristic gephyrin labeling and morphology of Renshaw cells. In the cat, fewer than half of the Renshaw cells (47%; n = 128) were double-labeled. In both species, occasional calbindin D28k-immunoreactive Renshaw cells were identified within motor nuclei in lamina IX. The distinctive immunolabeling of Renshaw cells allowed us to estimate that there are about 250 Renshaw cells in each ventral horn of the fourth lumbar segment of rat spinal cord, and about 750 cells per ventral horn in the L6 segment of the cat. We conclude that the functional properties of Renshaw cells, including their ability to fire action potentials at high rates, likely require specific homeostatic mechanisms including strong intracellular calcium buffering, the precise mechanisms of which may vary between species.