In the new human EndoC-beta H1 beta-cell line, a detailed analysis of the physiological characteristics was performed. This new human beta-cell line expressed all target structures on the gene and protein level, which are crucial for physiological function and insulin secretion induced by glucose and other secretagogues. Glucose influx measurements revealed an excellent uptake capacity of EndoC-beta H1 beta-cells by the Glut1 and Glut2 glucose transporters. A high expression level of glucokinase enabled efficient glucose phosphorylation, increasing the ATP/ADP ratio along with stimulation of insulin secretion in the physiological glucose concentration range. The EC50 value of glucose for insulin secretion was 10.3 mM. Mannoheptulose, a specific glucokinase inhibitor, blocked glucose-induced insulin secretion (GSIS). The nutrient insulin secretagogues L-leucine and 2-ketoisocaproate also stimulated insulin secretion, with a potentiating effect of L-glutamine. The Kir 6.2 potassium channel blocker glibenclamide and Bay K 8644, an opener of the voltage-sensitive Ca2+ channel significantly potentiated GSIS. Potentiation of GSIS by IBMX and forskolin went along with a strong stimulation of cAMP generation. In conclusion, the new human EndoC-beta H1 beta-cell line fully mirrors the analogous physiological characteristics of primary mouse, rat and human beta-cells. Thus, this new human EndoC-beta H1 beta-cell line is very well suited for physiological beta-cell studies. (C) 2015 Elsevier Inc. All rights reserved.