Ontogenetic appearances of glucagon, insulin and tyrosine hydroxylase (TH) were immunohistochemically investigated on developing pancreatic islets of rats. Glucagon immunoreactivity appeared first in some epithelial cells (g-cells) of the dorsal anlage of the pancreas on day 11.5 of gestation. On day 12.5, g-cells increased in number manufacturing the primitive islets, in which some cells appeared to be immunoreactive for insulin (i-cells) and about 40% of g-cells indicated also a slight immunoreactivity for insulin (g/i-cells). Afterwards, all the islet cells, especially g-cells, increased in number, and almost half of g-cells were g/i-cells. After day 16.5 of gestation, numerical increase of the cells with insulin immunoreactivity exceeded that of the cells with glucagon immunoreactivity, and about one fifth of g-cells were g/i-cells. After 20.5 days, however, no g/i cells were found. On day 16.5 of gestation, the immunoreactivity for TH appeared in occasional cells of the islets, but the cells did not show immunoreactivity for glucagon or insulin. It is concluded that the progenitor cells of the pancreatic islets appear to synthesize both glucagon and insulin by day 20.5 of gestation, but differentiate giving rise to mature A and B cells of adult islets afterward.