Recent predator demand and prey supply studies suggest that an annual daytime bottom trawl survey of Lake Superior underestimates prey fish biomass. A multiple-gear (acoustics, bottom trawl, and midwater trawl) nighttime survey has been recommended, but before abandoning a long-term daytime survey the effectiveness of night sampling of important prey species must be verified. We sampled three bottom depths (30, 60, and 120 m) at a Lake Superior site where the fish community included all commercially and ecologically important species. Day and night samples were collected within 48 h at all depths during eight different periods (one new and one full moon period during both early summer and late summer to early fall over 2 years). Biomass of demersal and benthic species was higher in night bottom trawl samples than in day bottom trawl samples. Night acoustic collections showed that pelagic fish typically occupied water cooler than 15°C and light levels less than 0.001 lx. Using biomass in night bottom trawls and acoustic biomass above the bottom trawl path, we calculated an index of acoustic detectability for each species. Ciscoes Coregonus artedi, kiyis C. kiyi, and rainbow smeltOsmerus mordax left the bottom at night, whereas bloaters C. hoyi stayed nearer the bottom. We compared the biomass of important prey species estimated with two survey types: day bottom trawls and night estimates of the entire water column (bottom trawl biomass plus acoustic biomass). The biomass of large ciscoes (>200 mm) was significantly greater when measured at night than when measured during daylight, but the differences for other sizes of important species did not vary significantly by survey type. Nighttime of late summer is a period when conditions for biomass estimation are largely invariant, and all important prey species can be sampled using a multiple-gear approach.