Many lactic acid bacteria produce diacetyl, which is a desirable aroma compound in some fermented dairy products. Strains or mutants of Lactococcus lactis subsp. lactis biovar. diacetylactis that are deficient in alpha-acetolactate decarboxylase are used in some food processes for their ability to produce large amounts of diacetyl. However, up until now, the use of alpha-acetolactate decarboxylase-deficient mutants of Streptococcus thermophilus for increased diacetyl production has not been evaluated. The objective of the present study was to devise a procedure for selecting spontaneous alpha-acetolactate decarboxylase-deficient mutants of S. thermophilus. We observed that in a chemically defined medium (CDM) containing alpha-ketobutyrate plus leucine, or alpha-ketobutyrate plus leucine plus isoleucine, the alpha-acetolactate decarboxylase-deficient mutant TIL865, obtained by directed mutagenesis, grew faster than its parent strain. This property was used for selecting spontaneous alpha-acetolactate decarboxylase-deficient mutants on agar plates. The resulting mutants were able to grow in milk, and their acidifying activity was slightly lower than that of the parent strain. Under partial anaerobic or aerobic conditions, they produced approximately three times more diacetyl than the parent strain. Such spontaneous mutants may be useful for increasing the diacetyl content of fermented milks whose production involves S. thermophilus strains.