Substrate preferences of juvenile, hatchery-reared lake sturgeon, Acipenser fulvescens, from a Wisconsin population were examined relative to water temperature and velocity, fish size and time of day. Given the choice of a sand, gravel, rock or smooth plastic bottom, all sturgeon were strongly attracted to the sand substrate. Water temperature did not affect substrate preference, however small sturgeon (13.5 cm mean FL) acclimated to 19°C were more active than those tested in 6°C water. Small sturgeon, under all conditions, were less sedentary in the early morning and late evening hours than during the daytime. Preference of small sturgeon for sand was slightly stronger when a current of 5 cm sec-1 was present. Substrate preference of larger sturgeon (24.3 cm mean FL) was not affected by any of the parameters examined. In general, hatchery-reared lake sturgeon showed similar behaviors (with respect to substrate selection) as those previously described for the same population in the wild, making it possible that substrate preferences have a genetic component and likely that hatchery rearing does not alter instinctive behavior.