AMONG the Eukaryota, the true fungi comprise four divisions (Chytridiomycota, Zymogomycota, Ascomycota and Basidio-mycota) that constitute a natural group which is thought to have diverged about 1 billion (109) years ago, believed also to be the time of divergence between metaphyta and metazoa lineages1. The endosymbionts responsible for the most prevalent plant root symbiosis, the vesicular-arbuscular mycorrhizae (VAM) or, more appropriately, arbuscular mycorrhizae, comprise 130 species of fungi classified in the Zygomycotina, order Glomales2-4. The arbuscular endomycorrhizae are considered to be ecologically important for most vascular plants5-8 in view of their beneficial effects on plant growth and survival. They are one of the few plant-fungus associations with a fossil record and may even have facilitated the origin of land flora. But the biochemical and genetic characterization of these microsymbionts has been hampered by the inability to grow them in pure culture. To investigate the origin and clarify the phylogenetic relationships of these organisms, we have sequenced ribosomal DNA genes from twelve species. Our phylogenetic analyses confirm the existence of three families within arbuscular fungi on the basis of morphological characters. We obtain approximate dates for the divergence of major branche's on the phylogenetic tree. These include an estimate for the origin of VAM-like fungi of 353-462 Myr ago, which is consistent with the hypothesis that VAM were instrumental in the colonization of land by ancient plants.