The identification of nuclear myosin I (NMI) has raised the possibility that myosin might have had an early functional role in the eukaryotic nucleus. To investigate this possibility, we examined the molecular evolution of the vertebrate myosin-I proteins. We found that myosin I has undergone at least five duplication events in the common ancestor of the vertebrates (vertebrate-specific duplications), leading to nine myosin-I vertebrate gene families, followed by two additional myosin-I duplication events in the lineage leading to modern fish. This expansion suggests a large-scale adaptive radiation in myosin-I function in an early phase of vertebrate evolution. The branching order of the evolutionary tree suggests that the functional role of NMI predates this expansion. More specifically, in the tunicate Ciona intestinalis, we found a myosin-I protein that localizes to the nucleus, but that branches on phylogenetic trees before the duplication that led to vertebrate myosin IC and myosin IH. This relationship suggests that the common ancestor of these three proteins encoded a nuclear isoform and that the localization of myosin I to the nucleus predates the origin of the vertebrates. Thus, a functional role for NMI appears to have been present at an early stage of animal evolution prior to the rise of both myosin IC and the vertebrates, as NMI was present in the last common ancestor of vertebrates and tunicates.