Fipronil and its transformation products are being increasingly detected in aquatic ecosystems as a result of widespread use. Fipronil is a chiral compound, and enantioselectivity may greatly impact its environmental fate and effects. In the present study, fipronil enantiomers were isolated and used to investigate the possibility of enantioselectivity in their acute toxicity to Japanese medaka (Oryzias latipes) and cytotoxicity to primary hepatocytes from rainbow trout (Oncorhynchus mykiss). Enantioselectivity was further evaluated in terms of fipronil degradation in sediments under aerobic and anaerobic conditions and in field-contaminated runoff water from urban watersheds. The 96-h median lethal concentrations to Japanese medaka were 94.2 (95% confidence interval [CI], 82.9-107.1), 98.3 (95% CI, 85.6-113.0), and 95.4 (95% CI, 74.7-121.9) gμg/L for the racemic, R-(−)-, and S-(+)-fipronil, respectively, suggesting absence of enantioselectivity. The 24-h median effect concentration of racemic fipronil to primary rainbow trout hepatocytes was 26.7 (95% CI, 25.6-27.9) μg/ml. In contrast, exposure of the cells to the S-(+)- and R-(−)-enantiomers resulted in a 19.7 and 7.8% reduction in cell viability, respectively, at the highest treatment concentrations (100 μg/ml), potentially indicating a greater-than-additive interaction between enantiomers. Under aerobic or slightly reduced conditions, biodegradation of fipronil in sediments was essentially nonstereoselective, with the enantiomeric fraction (EF) similar to racemic (EF = 0.5) after 168 d of incubation. However, EF decreased to as low as less than 0.1 following short incubations under anaerobic conditions, suggesting preferential degradation of S-(+)-fipronil in strongly reduced sediments. A survey of urban runoff samples consistently showed near-racemic EF, indicating fipronil degradation that was not enantioselective. Results suggest that site-specific characteristics are critical in accurately predicting fipronil fate and toxicity in the environment.