Elongation factor 1 alpha (EF-1 alpha) is a highly conserved ubiquitous protein involved in translation that has been suggested to have desirable properties for phylogenetic inference. To examine the utility of EF-1 alpha as a phylogenetic marker for eukaryotes, we studied three properties of EF-1 alpha trees: congruency with other phyogenetic markers, the impact of species sampling, and the degree of substitutional saturation occurring between taxa. Our analyses indicate that the EF-1 alpha tree is congruent with some other molecular phylogenies in identifying both the deepest branches and some recent relationships in the eukaryotic line of descent. However, the topology of the intermediate portion of the EF-1 alpha tree, occupied by most of the protist lineages, differs for different phylogenetic methods, and bootstrap values for branches are low. Most problematic in this region is the failure of all phylogenetic methods to resolve the monophyly of two higher-order protistan taxa, the Ciliophora and the Alveolata. JACKMONO analyses indicated that the impact of species sampling on bootstrap support for most internal nodes of the eukaryotic EF-1 alpha tree is extreme. Furthermore, a comparison of observed versus inferred numbers of substitutions indicates that multiple overlapping substitutions have occurred, especially on the branch separating the Eukaryota from the Archaebacteria, suggesting that the rooting of the eukaryotic tree on the diplomonad lineage should be treated with caution. Overall, these results suggest that the phylogenies obtained from EF-1 alpha are congruent with other molecular phylogenies in recovering the monophyly of groups such as the Metazoa, Fungi, Magnoliophyta, and Euglenozoa. However, the interrelationships between these and other protist lineages are not well resolved. This lack of resolution may result from the combined effects of poor taxonomic sampling, relatively few informative positions, large numbers of overlapping substitutions that obscure phylogenetic signal, and lineage-specific rate increases in the EF-1 alpha data set. It is also consistent with the nearly simultaneous diversification of major eukaryotic lineages implied by the "big-bang" hypothesis of eukaryote evolution.