Bacteria have always been considered ideal organisms for genetic analysis. While this is true for some model organisms, like Escherichia coli, Bacillus subtilis and, more recently, Lactococcus lactis, genetic analysis of other organisms is often prevented by lack of valuable tools, like vectors, transposons and methods for transformation, gene inactivation and random insertional mutagenesis. This is the case of the moderately thermophilic bacterium Streptococcus thermophilus, an organism that, in spite of its widespread use for food fermentations, is only poorly characterized. We report here an insertional mutagenesis system that allows efficient random mutagenesis, easy characterization of the interrupted genes and construction of stable null mutations. This may become a powerful S. thermophilus tool for both genetic analysis and construction of 'food-grade' mutants of this biotechnologically relevant microorganism.