Pancreatic βTC lines derived from insulinomas arising in transgenic mice expressing SV40 Tag under control of the insulin promoter manifest a differentiated β-cell phenotype and secrete insulin in response to glucose. Previously reported βTC lines respond to subphysiological extracellular glucose levels compared with normal β-cells. Recently, several βTC lines were developed with normal glucose-regulated insulin secretion from insulinomas obtained by breeding of the RIP-Tag transgene from the original C57BI/6 mouse strain into the C3HeB/FeJ strain. One of these βTC lines, βTC7, was characterized in detail. βTC7 cells express GLUT2 and have levels of glucokinase and hexokinase activity similar to those of normal islets. As a result these cells exhibit a normal glucose concentration dependency for glycolysis and insulin secretion, thus representing an accurate model of β-cell function. On continuous propagation in culture, βTC7 cells acquired a response to lower extracellular glucose levels. This change was associated with a fourfold increase in hexokinase activity, without significant changes in glucokinase activity and glucose uptake rates. These findings suggest an important role for glucose phosphorylation rates in regulation of the β-cell insulin secretory response to glucose.