Traditional views about the origin of eukaryotes and relationships between major “kingdoms” reflect interpretations of the fossil record and comparisons of phenotypic characters. This perspective is challenged by phylogenetic frameworks inferred from comparisons of macromolecular sequences which share a common ancestry. Similarities between ribosomal RNA genes demonstrate that instead of being relatively recent biological inventions, eukaryotes represent a discrete lineage that may be as old as the archaebacterial and eubacterial lines of descent. The diversity of protistan small subunit rRNA sequences exceeds that seen within the entire prokaryotic world. The earliest branching lineages include diplomonads, microsporidians, and tritrichomonads. Yet, other major protistan groups diverged relatively late in the evolutionary history of nucleated cells. Rather than being a concise evolutionary assemblage, the Protista should be regarded as a collection of paraphyletic lineages. In contrast, the Fungi, Plantae, and Animalia are independent monophyletic groupings. They originated nearly simultaneously during a relatively recent period characterized by a massive diversification of forms. This novel view of eukaryotic evolution suggests that a reliance upon large phenotypic differences in delineating kingdoms can obscure true genealogical relationships. Instead of dividing eukaryotes into four or more major divisions, they should be considered as a single kingdom that encompasses a progression of independently diverging lineages.