The cytotoxic effects of the herbicide alachlor were compared on rainbow trout gonadal RTG-2 and human neuroblastoma SH-SY5Y cell lines. The end points evaluated in both cells after 24, 48, and 72 h of exposure were total protein content (PC), lysosomal function, and mitochondrial's integrity by mitochondrial succinate dehydrogenase (SDH) activity. After 24 h, cytoplasmic membrane integrity by cytosolic lactate dehydrogenase (LDH) leakage and LDH intracellular activity were also studied. In addition, acetylcholinesterase activity (AChE) was quantified in SH-SY5Y cells. The possible biotransformation of alachlor by RTG-2 cells was investigated by analyzing the exposure culture medium by liquid chromatography-mass spectrometry. In RTG-2, EC50 values on PC, lysosomal function, and SDH activity after 24 h exposure ranged from 80 to 95 microM and decreased to approximately 40 microM for longer exposure time periods. SH-SY5Y cells were slightly more sensitive than RTG-2 cells, with EC50 values on PC and lysosomal function ranging from 87 to 75 microM at 24 h and decreasing to 47 microM and 34 microM at 72 h, respectively. AChE activity was increased, being the most sensitive marker for SH-SY5Y with an EC50 of 20 microM at 24 h. The metabolic enzyme SDH was stimulated in SH-SY5Y and reduced in RTG-2 cells. At the studied conditions, no metabolites of alachlor were detected in RTG-2 cultures. In conclusion, the proposed battery approach is an effective screening tool for the safety assessment of environmental contaminants as a complement to fish and animal toxicity procedures.