A monoclonal antibody against amino acids 224-234 of the gap junction protein connexin32 was found by immunohistochemistry to label subsurface cisterns (SSCs) in alpha-motoneurons of the rat (Yamamoto et al., 1990) and was used here to document by light (LM) and electron microscopy (EM) the appearance of immunoreactive SSCs in motoneurons of the rat and cat. This antibody and a polyclonal antibody against connexin32 labelled gap junctions in rat liver as well as SSCs in facial motoneurons. By LM, SSCs were seen as labelled puncta on motoneuronal perikarya and proximal dendrites. In the rat, they appeared to be present on all motoneurons at cranial and spinal levels, but varied considerably in size and number among motor nuclei. Labelled SSCs were the smallest and most sparse in motoneurons of the dorsal vagal motor nucleus, moderate in size and most numerous in the trochlear, oculomotor, and trigeminal motor nuclei, and largest though less densely distributed in spinal motoneurons. Dendrites were seen to contain SSCs for distances of up to 230 micron from their somal origin. Labelling within individual SSCs seen en face consisted of either numerous small puncta or linear arrays of immunoreactivity. By EM, labelled SSCs in the rat facial nucleus were always seen beneath a cluster of C-terminals. Immunolabelling was most dense in the space between the plasma membrane and SSC, which we define as the subsurface cisternal cleft. The SSCs were usually intermittently labelled along their length and exhibited a narrow luminal space ranging from 2 to 5 nm. On the basis of structural analogies between SSCs in neurons and the sacroplasmic reticulum terminal cistern/T-tubule complex in muscle, SSCs have previously been suggested to be important sites of calcium mobilization. The constant association of C-terminal with SSCs in motoneurons may represent a useful model in which to study SSC function as well as to investigate the possible presence of a connexinlike protein at regions of SSCs that form a narrow lumen similar to that at gap junctions.