Cells derived by trypsinization of neonatal golden hamster pancreas were cultured in modified Eagle's medium for 120 h in the presence of glucose (0.8 mg/ml) and for an additional 48 h in medium containing glucose (0.8 or 3.1 mg/ml) or tolbutamide (1,000 microg/ml) plus glucose (0.8 mg/ml). At day 7, cultures were stained differentially for light microscopy or examined by electron microscopy. Immunoreactive insulin (IRI) and immunoreactive glucagon (IRG) in the culture medium were measured by standard immunoassay procedures. Staining properties and ultrastructural appearance of cultured cells were comparable to those of the intact neonatal hamster pancreas. Cultures consisted predominantly of cells possessing aldehyde fuchsin positive (AF(+)) cytoplasmic granules resembling ultrastructurally those of the intact neonatal pancreatic beta cells and additionally, those of fibroblastoid, acinar, acino-insular, and aldehyde fuchsin negative (AF(-)) argyrophilic cells. IRI release rate by the cultured cells was increased in the presence of elevated glucose or tolbutamide which paralleled the loss of AF(+) granulation, but IRG release rate was suppressed by elevated glucose concentration. These findings indicate that these monolayer cultures consist of most of the cell types occurring in the neonatal pancreas, including endocrinologically competent islet cells.