As judged by autoradiographic criteria at the optical level, rat islet cells progressively accumulate 3H-glibenclamide over 1 to 30 min incubation at 24 degrees C. The labeled material associates with all endocrine cells, but is preferentially seen within B cells. The total uptake of 3H-glibenclamide (1.0 mumol/l) is little affected by the presence of unlabeled glibenclamide (0.2 mmol/l), but is significantly decreased at 4 degrees C (p less than 0.05). At the electron microscopic level, less than 15% of the autoradiographic grains are located at the B cell plasma membrane and 72-79% of the grains found over the cytoplasm are associated with insulin secretory granules. Such a pattern is observed both after short (1 min) or prolonged (30 min) incubation, and at 4 or 24 degrees C. It is proposed that the insulinotropic action of glibenclamide is not necessarily attributable to primary events located solely at the cell surface.