Six glucagon-secreting cell lines designated as In-R1G1,-G3,-G7,-G9,-G10, and-G11 were isolated from insulinoma cells (In-111-R1) by single cell cloning. A small amount of insulin was also detectable in the incubation medium when hormone secretion was stimulated by the addition of arginine or theophylline. These cell lines grew as monolayers and the population doubling times varied from 16.8 to 28.8 h. Karyologically these clones were aneuploid and the modes of chromosome numbers were 61 to 70. Electron microscopic examination of one of these clones showed that these cells contained moderately developed Golgi apparatus and a few secretory granules, which more or less resembled α-cell granules. By gell filtration study of the incubation medium, glucagon and glucagonlike material were eluted. The molecular weight of the latter was approximately 9000, which suggested the concomitant secretion of rpoglucagon into the medium. The levels of secreted glucagon in basal state were 0.3 to 3.0 ng/10⁶ cells/2 h. Glucagon secretion was markedly enhanced in the presence of amino acids. Glucagon secretion increased slightly in the presence of high concentration of glucose in Hanks' balanced salt solution; however it was not affected by the varying concentrations of glucose when the cells were incubated in complete media with amino acids. Glucagon secretion was also stimulated by the addition of theophylline. These clonal cell lines seem to provide a useful tool for investigating the mechanism of glucagon secretion.