A series of experiments were performed with laboratory scale activated sludge units to determine the effects of various environmental conditions on the ATP content of activated sludge mixed liquor. ATP was determined by the firefly luciferin-luciferase reaction. The effect of abrupt temperature changes up to about 37°C was small. Anoxia reduced the ATP content by about 40%, but an immediate return to the original level was noted when sludge was reaerated. Complex patterns of ATP response were found for substrate (milk solids) additions and for additions of various inorganic toxicants. ATP responses to toxic heavy metals are rapid and sensitive, and ATP appears to be a good measure of sludge viability and activity.