The aim of this study was to define when an insulin-positive cell becomes functionalin vivoand starts to exocytose insulin in a regulated nutrient-dependent manner. Insulin-positive cells appear in embryonic life (midgestation) and complete their maturation, presumably around birth. In order to work with embryonic and newborn endocrine pancreas, we used organotypic slices. The mouse embryonic pancreas slices show high basal insulin release that is not further elevated by high glucose levels. Despite the presence of functional voltage-activated ion channels, the cells are not electrically active in the presence of secretagogues. At birth, the high basal insulin release drops and, after postnatal day 2, the insulin-positive cells show both adult-like bursting electrical activity and hormone release induced by high glucose levels. These properties allowed us to define them as beta cells. Despite the apparent stability of the transcription factor profile reported in insulin-positive cells during late-embryonic life, functional beta cells appear only 2 days after birth.