We investigated the guidance efficiency of fish screens for the protection of emigrating Pacific LampreyEntosphenus tridentatusand Western River Lamprey (also known as River Lamprey)Lampetra ayresiiin a series of experimental trials. All trials were conducted at the Tracy Fish Collection Facility, located in the Sacramento–San Joaquin River Estuary at the entrance to one of the world’s largest surface water diversions. Using 1,200 lamprey macrophthalmia, we tested for the effect of screen type, time of day, and channel water velocity to guide their swimming behavior to avoid entrainment. We found overwhelming evidence for an effect of screen type on efficiency, whereby all lampreys were successfully guided to a holding tank when a vertical traveling screen was used. This was likely due to the small pore size of the screen relative to lamprey sizes. In contrast, the efficiency of louvers, a behavioral screen designed for salmonids, varied by the interaction of time of day and channel velocity. During nighttime, when lamprey typically emigrate, louver guidance efficiency ranged from 21% (95% CI, 14–30%) to 24% (95% CI, 16–34%). These results were applied to estimate the probability for salvage of lamprey macrophthalmia at the Tracy Fish Collection Facility, which includes a two-stage fish screen design. Between 1957 and 2014, we estimated that 94–96% of the lampreys that were entrained in the export flows were lost and not returned to the delta. However, the probability for fish loss was reduced in 2014 when the secondary louver was replaced with a vertical traveling screen. Our results suggest that lamprey macrophthalmia entrainment into the canals will be eliminated at the Tracy Fish Collection Facility if the primary screen is converted to vertical traveling screen. Surface water diversions may represent a substantial threat to regional metapopulations of anadromous lamprey species worldwide, and screening approaches applied to other fish species such as salmonids may not be protective of lampreys. Received June 7, 2016; accepted August 23, 2016Published online December 20, 2016 [ABSTRACT FROM AUTHOR]