The essential GroEL proteins represent a subset of molecular chaperones ubiquitously distributed among species of the eubacterial lineage, as well as in eukaryote organelles. We employed these highly conserved proteins to infer eubacterial phylogenies. GroEL from the species analyzed clustered in distinct groups in evolutionary trees drawn by either the distance or the parsimony method, which were in general agreement with those found by 16S rRNA comparisons (i.e., proteobacteria, chlamydiae, bacteroids, spirochetes, firmicutes [gram-positive bacteria], and cyanobacteria-chloroplasts). Moreover, the analysis indicated specific relationships between some of the aforementioned groups which appeared not to be clearly defined or controversial in rRNA-based phylogenetic studies. For instance, a monophyletic origin for the low-G+C and high-G+C subgroups among the firmicutes, as well as their specific relationship to the cyanobacteria-chloroplasts, was inferred. The general observations suggest that GroEL proteins provide valuable evolutionary tools for defining evolutionary relationships among the eubacterial lineage of life.