Whereas JUUL electronic cigarettes (ECs) have captured the majority of the EC market, with a large fraction of their sales going to adolescents, little is known about their cytotoxicity and potential effects on health. The purpose of this study was to determine flavor chemical and nicotine concentrations in the eight currently marketed prefilled JUUL EC cartridges ("pods") and to evaluate the cytotoxicity of the different variants (e.g., "Cool Mint" and "Crème Brulee") using in vitro assays. Nicotine and flavor chemicals were analyzed using gas chromatography-mass spectrometry in pod fluid before and after vaping and in the corresponding aerosols. 59 flavor chemicals were identified in JUUL pod fluids, and 3 were >1 mg/mL. Duplicate pods were similar in flavor chemical composition and concentration. Nicotine concentrations (average 60.9 mg/mL) were significantly higher than those of any EC products we have previously analyzed. The transfer efficiency of individual flavor chemicals that were >1 mg/mL and nicotine from the pod fluid into aerosols was generally 35-80%. All pod fluids were cytotoxic at a 1:10 dilution (10%) in the MTT and neutral red uptake assays when tested with BEAS-2B lung epithelial cells. Most aerosols were cytotoxic in these assays at concentrations between 0.2 and 1.8%. The cytotoxicity of collected aerosol materials was highly correlated with nicotine and ethyl maltol concentrations and moderately to weakly correlated with total flavor chemical concentration and menthol concentration. Our study demonstrates that (1) some JUUL flavor pods have sufficiently high concentrations of flavor chemicals that may make them attractive to youth and (2) the concentrations of nicotine and some flavor chemicals (e.g., ethyl maltol) are high enough to be cytotoxic in acute in vitro assays, emphasizing the need to determine if JUUL products will lead to adverse health effects with chronic use.