The calcium/calmodulin-dependent protein phosphatase calcineurin was localized at the light microscopic level in the rat hindbrain and spinal cord by using an antibody against the alpha-isoform of the catalytic subunit. Calcineurin was highly concentrated in axons, dendrites, and cell bodies of a subpopulation of alpha-motoneurons in hindbrain motor nuclei and the lateral motor column along the length of the spinal cord. These calcineurin-positive alpha-motoneurons appeared to be randomly distributed and represented approximately 25% of the total alpha-motoneuron pool in the motor trigeminal nucleus and the spinal cord lateral motor column. Within the facial nucleus, calcineurin-containing motoneurons were present in the medial and dorsal subdivision but not in the lateral and intermediate subdivision. In addition to the enrichment in motoneurons, calcineurin was enriched in cells of the superficial laminae of the spinal cord dorsal horn and its extension into the medulla, the caudal spinal trigeminal nucleus. Axonal staining in the white matter of the spinal cord was generally weak, except in the dorsolateral funiculus, where strongly calcineurin-positive axons formed a putative ascending tract that appeared to terminate uncrossed in the caudal lateral reticular nucleus of the medulla. This tract may originate from calcineurin-positive cells in the dorsolateral funiculus. We also compared the distribution of calcineurin with calcium/calmodulin-dependent kinase II in the spinal cord and found that the kinase is more widely expressed. Thus, calcineurin is highly restricted to a few locations in the hindbrain and spinal cord. Selective staining in facial subnuclei that innervate phasically active muscles suggests that calcineurin-positive motoneurons represent a subset of alpha-motoneurons innervating a metabolic subtype of muscle fibers, possibly fast-twitch fibers.