ABSTRACTOne-dimensional search is an essential step in DNA target recognition. Theoretical studies have suggested that the sequence dependence of one-dimensional diffusion can help resolve the competing demands of fast search and high target affinity, a conflict known as the speed-selectivity paradox. The resolution requires that the diffusion energy landscape is correlated with the underlying specific binding energies. In this work, we report observations of one-dimensional search by QD labeled EcoRI. Our data supports the view that proteins search DNA via rotation coupled sliding over a corrugated energy landscape. We observed that while EcoRI primarily slides along DNA at low salt concentrations, at higher concentrations its diffusion is a combination of sliding and hopping. We also observed long-lived pauses at genomic star sites which differ by a single nucleotide from the target sequence. To reconcile these observations with prior biochemical and structural data, we propose a model of search in which the protein slides over a sequence independent energy landscape during fast search, but rapidly interconverts with a “hemi-specific” binding mode in which a half site is probed. This half site interaction stabilizes the transition to a fully specific mode of binding which can then lead to target recognition.