DNA sequences of the gene tufA, encoding elongation factor Tu, were determined from five cyanobacteria and 21 plastids. Three were full length (ca. 1230 bp) sequences from cloned DNA, and 23 were partial (ca. 740 bp) sequences from PCR fragments. These sequences were aligned with sequences available from the literature, creating a data set of 56 tufA sequences of eubacterial or plastid origin. Phylogenetic analysis was performed on inferred amino acid sequences with parsimony and neighbor joining techniques, and on first and second position nucleotide sequences with maximum likelihood, and bootstrapping was performed with each method. Trees determined by the three methods were highly congruent with respect to well supported nodes. All examined plastids, including those of green and red algae, chromophytes, and Cyanophora paradoxa, cluster strongly with the cyanobacteria in all analyses. A cyanobacterial origin of all plastids confirms phylogenetic analyses of 16S rRNA and atpB sequences, but conflicts with those of rbcL and rbcS sequences. This discrepancy may be attributable to an ancient gene transfer of the rubisco operon in an ancestor of red algae and chromophytes. Maximum likelihood analysis also provides some support for a monophyletic origin of all plastids, while neighbor joining and parsimony analyses showed cyanobacteria and red, brown, and green plastid lineages as an unresolved polytomy. These tufA analyses also provide a broad perspective on eubacterial evolution, and in conjunction with published rRNA trees, point to at least two major radiations within eubacteria and their descendants: one of many eubacterial phyla, a second of cyanobacteria, and possibly a third radiation early in plastid evolution.