The abandoned São Domingos mine (Portugal) offers a pH and metal gradient of acid mine drainage (AMD), and is an ideal model for ecotoxicological studies. Short-term 24-h bioassays with water from the AMD (pH 3.3, 4.4, and 5.5, and control) were performed with fourth instars of Chironomus in the laboratory and in situ (AMD at pH 5.5) in artificial flow-through channels. This was compared to reference water acidified to the respective pH values (acid only). Behavioral stress responses were monitored online with the Multispecies Freshwater Biomonitor® (MFB). The exposure to AMD and acid only was in the sublethal range (mortality 0–20%). The use of MFB chambers did not affect survival. Stress behavior of Chironomus consisted mainly of decreased locomotory activity in AMD and increased activity in acid-only tests, indicating that the metals in the AMD played a role as stress factor. Field exposure in the AMD mixing zone (pH 5.5) generated similar activity as in the corresponding laboratory exposure.