|As fish cell cultures continue to be explored as alternatives to whole fish for evaluating the toxicity of environment chemicals, technical issues have emerged that influence results and thus need to be understood and standardized. These include carrier solvents, dosing protocols, exposure vessel, exposure media, viability endpoints, and cell lines. Some of these factors have been explored in this thesis for eight reactive contaminants exhibiting varied physicochemical properties using the rainbow trout cell lines RTgill-W1 and RTL-W1. Sodium dodecyl sulphate (SDS) was used as a reference (control) chemical. Cell viability was evaluated with alamar Blue, carboxyfluoroscein diacetate acetoxymethyl ester and neutral red as measures respectively of metabolic activity, plasma membrane integrity, and lysosomal function. Experimental in vitro EC50 values were compared to 1) pre-existing in vivo LC50s from the fathead minnow database and 2) pre-existing in vitro EC50s from the Halle database. Results point to good in vitro/in vivo correlations for menadione, dichlorophene, hexachlorophene, and acrolein. Poor correlations were observed for allyl alcohol, 4-fluoroaniline, acetaldehyde, and 2,3-dimethyl-1,3-butadiene due to a combination of solubility and volatility problems. Overall, the results suggest that the impact of different technical approaches on the evaluation of acute toxicity in vitro depends very much on the chemical class being investigated and less on the characteristics of the cell line. The in vitro cytotoxicity of reactive chemicals is challenging due to the nature of the chemicals’ physicochemical properties. Further improving the in vitro toxicity of reactive chemicals is a prerequisite for the ultimate goal of using fish cell cultures as acceptable, standard alternatives to the use of fish acute lethality assays.