Eastern sand darter were found exclusively associated with sand substrates in the field and in 90\% of observations made on individuals in an artificial stream under controlled conditions. In the study stream, the Mettawee River, other physical habitat variables, such as, water velocity, depth or distance from bank, had no detectable effect on distribution or substrate use. In artificial streams, neither water velocity nor depth affected substrate use. No diel changes in substrate use were noted in laboratory experiments. Distribution of eastern sand darter was not associated with the distribution of other fishes present in study stream. Few temperate stream fishes are as strongly associated with a particular habitat variable as is Ammocrypta pellucida which is important to note when developing management strategies. The number of streams in which this species occurs has declined during the last half century. These declines may reflect changes in habitat.