The effects of glucose on insulin gene expression and proinsulin biosynthesis, processing, and secretion were studied in mouse beta TC3 cells, an established insulinoma cell line derived from transgenic mice carrying a hybrid insulin promotor-simian virus-40 tumor antigen. The level of insulin mRNA was maintained at high levels by culture in 11 mM glucose, but essentially disappeared after 48 h of culture without glucose. The rate of insulin biosynthesis in beta TC3 cells was also dependent on glucose concentration over periods of 24 or 48 h (but not during 3 h) of stimulation. Insulin biosynthesis decreased about 50% after 24 h and about 85% after 48 h of incubation without glucose. When beta TC3 cells were incubated without glucose for 48 h, the rate of conversion of proinsulin to insulin was decreased compared to that at 11 mM glucose. Insulin secretion was sustained by medium glucose and also exhibited a much lower threshold for maximal stimulation; 2-deoxyglucose uptake decreased about 50% after 48 h of incubation without glucose. Studies on the secretion of newly synthesized proinsulin/insulin revealed that up to 80% of the total cellular pool of labeled proinsulin was released during a 60-min chase compared to only 10% of labeled insulin. The release of immunoreactive insulin (IRI) during the chase period was stimulated by forskolin and phorbol-12-myristate-13-acetate 1.6- and 10-fold, respectively. However, the release of newly synthesized proinsulin was insensitive to these secretagogues. It is concluded that 1) as in normal islets, glucose influences the steady state levels of proinsulin mRNA in beta TC3 cells; 2) the rate of proinsulin biosynthesis reflects only the level of insulin mRNA; translational control is absent; 3) cellular conversion of proinsulin to insulin is up-regulated by glucose as in normal rat islets; 4) newly synthesized proinsulin is released predominantly via a constitutive, rather than a regulated pathway, in contrast to normal beta-cells.