Abstract Seventy-four wine strains of the Saccharomycesgenus were tested to study their oenological characteristics and enzymatic activities. These strains were isolated from spontaneous microbiota taken from cellars in the Valdepeñas region (Spain) and identified by contour homogeneous electric field (CHEF) technique. When Cluster Analysis was applied to oenological properties, only SH 2production, tolerance to 12 0of ethanol and synthesis of killer toxin contributed to the distribution of the strains in the different statistical groups. All the strains possessed esterase activity and some also showed protease and pectinase activities. None of them had β-D-glucosidase activity.