The present study evaluates the development and functional properties of beta cells differentiated in vitro. The authors have previously demonstrated that when E12.5 rat pancreatic rudiments are cultured in vitro in the absence of mesenchyme, the majority of the epithelial cells differentiate into endocrine beta cells. Thus, depletion of the mesenchyme provokes the expansion of endocrine tissue at the expense of exocrine tissue. The potential use of this procedure for the production of beta cells led the authors to characterize the beta cells differentiated in this model and to compare their properties with those of the endocrine cells of the embryonic and adult pancreas. This study shows that the beta cells that differentiate in vitro in the absence of mesenchyme express the homeodomain protein Nkx6.1, a transcription factor that is characteristic of adult mature beta cells. Further, electron microscopy analysis shows that these beta cells are highly granulated, and the ultrastructural analysis of the granules shows that they are characteristic of mature beta cells. The maturity of these granules was confirmed by a double-immunofluorescence study that demonstrated that Rab3A and SNAP-25, two proteins associated with the secretory pathway of insulin, are strongly expressed. Finally, the maturity of the differentiated beta cells in this model was confirmed when the cells responded to stimulation with 16 mM glucose by a 5-fold increase in insulin release. The authors conclude that the beta cells differentiated in vitro from rat embryonic pancreatic rudiments devoid of mesenchyme are mature beta cells.