Autoimmune destruction of β cells in the pancreas leads to type I, or insulin dependent diabetes mellitus (IDDM), through the loss of endogenous insulin production capacity. This paper describes an attempt to generate ‘artificial’β cells using the fibroblast cell line BHK21. Stable transfectants expressing the human preproinsulin (PPI) gene were isolated and characterised. The resulting clone selected for further analysis (BHK-PPI-C16) was capable of secreting 0.12 pmol proinsulin/hr/105 cells and maintained a steady cellular proinsulin content of 0.36 ± 0.04 pmol l−1. There was no processing of the proinsulin to mature insulin. The cells were unresponsive to glucose but there was increased proinsulin secretion in the presence of agents that stimulated formation of intracellular cAMP. Transfection of cDNAs for the key elements of the glucose sensing apparatus (GLUT2 and glucokinase) led to a subphysiological stimulation of secretion when glucokinase was transfected alone while there was a complete loss of insulin secretion when both components were overexpressed. The deleterious effect on proinsulin secretion observed upon co-expression of the glucose sensing genes may have implications for applications requiring multigene expression in BHK21 cells.