Aims: To improve a method for determining beta-glucosidase activity and to apply it in yeasts isolated from wine ecosystems from 'La Mancha' region and to know its cellular location. Methods and Results: A total of 82 wine yeasts were identified (PCR/RFLP) and evaluated for their beta-glucosidase activity. First, they were qualitatively evaluated by growth on YNB cellobiose, the activity was quantified using different culture media, under aerobic and anaerobic conditions and cells after 24-72 h of growth. To study the location activity, five fractions were obtained (supernatant, whole cell, cell wall, cytosol and cell membrane). The enzymatic assays were optimized, being: growth in YP cellobiose for 72 h in aeration conditions and, after cell removing, enzyme analysis with 128 g l(-1) of cellobiose as substrate, for 30 min at 30 degrees C. The genus that displayed the greatest activity were Pichia, Hanseniaspora and Rhodotorula, and the activity was intracellular. Conclusions: The study showed that beta-glucosidase activity was induced by the carbon source and was aerobic dependent. The non-Saccharomyces species displayed the greatest activity, which was intracellular and strain-dependent. Significance and Impact of the Study: This study developed a reliable method for screening beta-glucosidase activity in yeasts isolated from wine ecosystems. This activity is very important in the release of monoterpenols from glycoside precursors for the enhancement of wine aromas.