Ray-finned fishes make up half of all living vertebrate species. Nearly all ray-finned fishes are teleosts, which include most commercially important fish species, several model organisms for genomics and developmental biology, and the dominant component of marine and freshwater vertebrate faunas. Despite the economic and scientific importance of ray-finned fishes, the lack of a single comprehensive phylogeny with corresponding divergence-time estimates has limited our understanding of the evolution and diversification of this radiation. Our analyses, which use multiple nuclear gene sequences in conjunction with 36 fossil age constraints, result in a well-supported phylogeny of all major rayfinned fish lineages and molecular age estimates that are generally consistent with the fossil record. This phylogeny informs three longstanding problems: specifically identifying elopomorphs (eels and tarpons) as the sister lineage of all other teleosts, providing a unique hypothesis on the radiation of early euteleosts, and offering a promising strategy for resolution of the "bush at the top of the tree" that includes percomorphs and other spiny-finned teleosts. Contrasting our divergence time estimates with studies using a single nuclear gene or whole mitochondrial genomes, we find that the former underestimates ages of the oldest ray-finned fish divergences, but the latter dramatically overestimates ages for derived teleost lineages. Our time-calibrated phylogeny reveals that much of the diversification leading to extant groups of teleosts occurred between the late Mesozoic and early Cenozoic, identifying this period as the "Second Age of Fishes."Actinopterygii | molecular clock | species tree | Teleostei | Percomorpha R ay-finned fishes (Actinopterygii) are one of the most successful radiations in the long evolutionary history of vertebrates, yet despite the rapid progress toward reconstructing the Vertebrate Tree of Life, only 5% of the ray-finned fish phylogeny is resolved with strong support (1). Actinopterygii contains more than 30,000 species (2), with all but 50 being teleosts (3). Compared with other large vertebrate radiations, such as mammals (4) or birds (5), a general consensus on the phylogenetic relationships and timing of diversification among the major actinopterygian and teleost lineages is lacking (3,6,7). This uncertainty about relationships has prevented the development of a comprehensive time-calibrated phylogeny of ray-finned fishes, which is necessary to understand macroevolutionary processes that underlie their diversity.Most working concepts of actinopterygian relationships are based on morphological data (6, 8), and unlike other clades of vertebrates, there has been no comprehensive effort to resolve the phylogeny of actinopterygians and teleosts using molecular data that sample multiple nuclear genes and include taxa that span the major lineages. Despite the long history of using morphological data in the phylogenetics of ray-finned fishes, there are several areas of uncertainty and disagreement...