In the rat pineal organ numerous stellate cells exhibited intense immunoreactivity for calcium-binding spot 35 protein. Because of their peculiar shape and ultrastructure, identical to those of intrapineal S-100-immunoreactive cells, the spot 35-immunoreactive stellate cells were identified as the interstitial cells. The comparison of the morphology and population density of spot 35-, S-100-, and GFAP (glial fibrillar acidic protein)-immunoreactive cells, suggests that spot 35-immunoreactive cells represent a major subpopulation of the interstitial cells, all of which are S-100-immunoreactive and generally considered to be of glial nature, while GFAP-immunoreactive cells represent a minor subpopulation of the interstitial cells located in the proximal part close to the pineal stalk. This is the first report describing the occurrence of the calcium-binding protein in cells of glial nature. © 1990 Chapman and Hall Ltd.