2,3,3,3-Tetrafluoropropene (HFO-1234yf) is a non-ozone-depleting fluorocarbon replacement with a low global warming potential which has been developed as refrigerant. The biotransformation of HFO-1234yf was investigated after inhalation exposure. Male Sprague–Dawley rats were exposed to air containing 2000, 10,000, or 50,000 ppm HFO-1234yf for 6 h and male B6C3F1 mice were exposed to 50,000 ppm HFO-1234yf for 3.5 h in a dynamic exposure chamber (n=5/concentration). After the end of the exposure, animals were individually housed in metabolic cages and urines were collected at 6 or 12-hour intervals for 48 h. For metabolite identification, urine samples were analyzed by 1H-coupled and decoupled 19F-NMR and by LC/MS-MS or GC/MS. Metabolites were identified by 19F-NMR chemical shifts, signal multiplicity, 1H–19F coupling constants and by comparison with synthetic reference compounds. In all urine samples, the predominant metabolites were two diastereomers of N-acetyl-S-(3,3,3-trifluoro-2-hydroxy-propyl)-l-cysteine. In 19F-NMR, the signal intensity of these metabolites represented more than 85% (50,000 ppm) of total 19F related signals in the urine samples. Trifluoroacetic acid, 3,3,3-trifluorolactic acid, 3,3,3-trifluoro-1-hydroxyacetone, 3,3,3-trifluoroacetone and 3,3,3-trifluoro-1,2-dihydroxypropane were present as minor metabolites. Quantification of N-acetyl-S-(3,3,3-trifluoro-2-hydroxy-propyl)-l-cysteine by LC/MS-MS showed that most of this metabolite (90%) was excreted within 18 h after the end of exposure (t1/2 app. 6 h). In rats, the recovery of N-acetyl-S-(3,3,3-trifluoro-2-hydroxy-propyl)-l-cysteine excreted within 48 h in urine was determined as 0.30±0.03, 0.63±0.16, and 2.43±0.86 μmol at 2000, 10,000 and 50,000 ppm, respectively suggesting only a low extent (<<1% of dose received) of biotransformation of HFO-1234yf. In mice, the recovery of this metabolite was 1.774±0.4 μmol. Metabolites identified after in vitro incubations of HFO-1234yf in liver microsomes from rat, rabbit, and human support the metabolic pathways of HFO-1234yf revealed in vivo. The obtained results suggest that HFO-1234yf is subjected to a typical biotransformation reaction for haloolefins, likely by a cytochrome P450 2E1-catalyzed formation of 2,3,3,3-tetrafluoroepoxypropane at low rates, followed by glutathione conjugation or hydrolytic ring opening.