The phylogenetic relationships between species of yeasts assigned to the Saccharomyces sensu stricto group, which includes Saccharomyces cerevisiae and Saccharomyces bayanus, were studied together with Saccharomyces pastorianus and Saccharomyces paradoxus. The experimental approaches used were RFLP analysis of the PCR-amplified rDNA internal transcribed spacer (ITS) and intergenic spacer, and total ITS sequence analysis. Both RFLP and sequence analyses gave fairly similar results. The gene trees generated with either of the two data sets showed the distribution of the yeasts into two major, well-separated, phylogenetic clusters called 'cerevisiae' and 'bayanus'. The 'cerevisiae' cluster included the S. cerevisiae type strain, together with most of the species (16 out of 23), whereas the 'bayanus' cluster included the remaining seven type strains. Therefore, analysis of rDNA sequences confirmed S. cerevisiae and S. bayanus as two well-defined taxa. However, S. pastorianus and S. paradoxus, the two other usually accepted taxa of the now-defined Saccharomyces sensu stricto complex, could not be clearly separated from S. bayanus and S. cerevisiae, respectively. However, in both PCR-RFLP and ITS sequence analyses, S. paradoxus had the outermost position in the 'cerevisiae' cluster. PCR-RFLP analysis of the ribosomal spacer sequences was also carried out on 26 Saccharomyces strains isolated in various wine-growing regions of France in an attempt to clarify their positions in the Saccharomyces phylogenetic tree. Compared to the diversity of the Saccharomyces type strains, less genetic diversity was detected among these yeasts and several of them exhibited identical RFLP patterns. Most of the wine yeast strains (16 out of 26) were closely related to each other and were found within the 'cerevisiae' cluster. The remaining 10 wine yeast strains branched within the 'bayanus' cluster. PCR-RFLP analysis of ribosomal spacer sequences thus appears to be a useful and appropriate method for the correct characterization of Saccharomyces yeast strains used in food processing.