Introduction: [18F]FMISO, the widely used positron emission tomography (PET) hypoxia tracer, is a chiralcompound clinically used as a racemic mixture. The purpose of this study was to synthesize theindividual (R)- and the (S)- enantiomers of [18F]FMISO and compare their PET imaging characteristics.Methods: The radiosynthesis of enantiopure (R)- and (S[18F]FMISO was based on Co(salen) (N,N'-bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexanediaminocobalt)-mediated opening of enantiopure epoxideswith [18F]HF. The uptake and clearance of the individual [18F]FMISO antipodes were investigated usingmicro-PET/CT imaging performed on mice bearing FaDu tumors. Image-derived biodistribution wasobtained from micro-PET/CT scans performed at 1 and 3 hours post injection (p.i.). In addition, theuptake patterns of each enantiomer were observed using two-hour dynamic micro-PET/CT scans andthe time-activity curves from different organs were compared. Results: The individual (R)- and (S)-[18F]FMISO enantiomers were synthesized in one step with highenantiomeric excess (ee) > 99% and radiochemical purity > 97% using custom-made automationmodule. The dynamic micro-PET/CT scanning revealed a faster initial uptake of the (R)-[18F]FMISOenantiomer in tumor and muscle tissues, however the difference became progressively smaller withtime. The tumor-to-muscle (T/M) and tumor-to-liver (T/L) ratios remained nearly identical for the (R)-and (S)-forms at all time points. The micro-PET/CT imaging at 1 and 3 hours p.i. did not show anysignificant enantioselective tissue uptake. Conclusions: Although the (R)-enantiomer of [18F]FMISO demonstrated a somewhat faster initial tumorand muscle uptake no significant enantioselective tissue uptake was observed at later time points. TheT/M- and T/L- ratios for the (R)- and (S)-forms were the same within the experimental error at all times.Therefore, the use of enantiopure [18F]FMISO is unlikely to present any practical clinical benefit for PET imaging.