Spot 35 protein is identical to rat brain calbindin, as based on the complete homology of their nucleotide sequence, and is named here as spot 35-calbindin. The immunoreactivity for this protein was first detected on embryonic Day 16 (E16) in the anterior pituitary anlages. As the development proceeded, spot 35-immunoreactive cells increased in number in the pars distalis, especially in its antero-ventral portion, and also pars tuberalis. No sex difference was noticed in the population density of the immunoreactive cells until postnatal Week 3. At postnatal Week 5 and thereafter through adulthood, spot 35-immunoreactive cells were much more numerous in the male anterior pituitary than in the female organ. All immunopositive cells for spot 35-calbindin contained secretory granules, and no folliculo-stellate cells were immunopositive. Any of the anterior pituitary hormones including ACTH, GH, LH, PRL and TSH could be simultaneously expressed with spot 35-calbindin in the granulated cells. Based on the ultrastructure of the secretory granules, spot 35-immunoreactive cells were classified into four types: cells containing round granules, 350 nm; those containing granules of irregular contour larger than 350 nm; those containing small granules, up to 150 nm; and those containing a mixture of the round large and small granules. Cells containing large round granules were dominant in males, while cells containing small granules were dominant in females. The functional significance of spot 35-calbindin in the anterior pituitary is briefly discussed.