Although the food industry has risen to the formulation challenges associated with removing gluten from the dough, and a number of gluten-free (GF) products are now commercially available on the market, many GF breads formulations are frequently based on pure starches and still result in low technological and nutritional quality, dry crumb and poor overall mouthfeel and flavor of the final breads (Mariotti et al., 2013). Sourdough is the foremost fermentation used for baking purposes and it has been proven to be ideal for improving the texture, palatability, aroma, shelf-life and nutritional value of wheat and rye breads. Even if these effects have been extensively studied and well described for the traditional baking, little is known about the role of sourdough in GF baking (Moroni et al., 2009). In the last years, however, some studies evidenced various improvements in GF bread volume, crumb structure, flavor and shelf-life (mold-free) when a sourdough was used as leavening agent. All these benefits are mainly due to the starter cultures used and to the organic acids and aminoacids released during the leavening process. In this contest, the aim of this study was the set up of a GF sourdough from selected bacteria and yeasts isolated from a wheat-based sourdough. In particular, the microorganisms of interest (Candida humilis and Lactobacillus sanfranciscensis) were isolated from a traditional sourdough used for the production of Panettone (a well-known Italian sweet baked product). The isolated strains were inoculated (105 CFU/g C. humilis, 107 CFU/g Lb. sanfranciscensis) into a GF matrix, containing corn starch, rice flour, pea isolate, hydrocolloids and fiber as main ingredients (the optimized recipe comes from previous studies). The mass was fermented at 25°C for 24h. The GF inoculum was then added (10% integration level) to another GF matrix, very similar to the previous one, to produce the GF sourdough. This starter (called ‘mother dough’) was constantly and continuously propagated (10 refreshments covering a period of 30 days), and its properties were regularly monitored, particularly in terms of number and type of microorganisms, dough consistency, dough titratable acidity and capability to produce and retain CO2 (rheofermentographic test). During the fermentation process, the pH was continuously recorded and the leavening phase was considered complete when the values were equal to 3.8-4.0. A stable association between microorganisms, measured both in terms of microbiological population (e.g. number of microorganisms, type of microorganisms, ratio between yeasts and bacteria) and technological properties (e.g. amount of water added to the dough to reach the desired consistency, time required to obtain the full sourdough, CO2 production and retention volumes, titratable acidity etc.) of the GF sourdough was obtained just after the second refreshment; all these properties remained constant and satisfactory from here onwards, thus attesting that a GF sourdough had effectively been developed. Only at this point some bread-making trials were performed, in order to compare the properties and quality of GF breads obtained using the developed GF-sourdough, the compressed yeast (Saccharomyces cerevisiae) or their mixture, as leavening agents (see: Gluten-free bread. II. Compressed yeast versus sourdough).