OBJECTIVES: The purpose of this study was to systematically assess the optimal b-value and reconstruction parameters for DTI and fiber tractography of the median nerve at 3.0T. METHODS: Local ethical board approved study with 45 healthy volunteers (15 men, 30 women; mean age, 41±3.4 years) who underwent DTI of the right wrist at 3.0T. A single-shot echo-planar-imaging sequence (TR/TE 10123/40ms) was acquired at four different b-values (800, 1000, 1200, and 1400s/mm(2)). Two independent readers performed post processing and fiber-tractography. Fractional anisotropy (FA) maps were calculated. Fiber tracts of the median nerve were generated using four different algorithms containing different FA thresholds and different angulation tolerances. Data were evaluated quantitatively and qualitatively. RESULTS: Tracking algorithms using a minimum FA threshold of 0.2 and a maximum angulation of 10° were significantly better than other algorithms. Fiber tractography generated significantly longer fibers in DTI acquisitions with higher b-values (1200 and 1400s/mm(2) versus 800s/mm(2); p<0.001). The overall quality of fiber tractography was best at a b-value of 1200s/mm(2) (p<0.001). CONCLUSIONS: In conclusion, our results indicate use of b-values between 1000 and 1400s/mm(2) for DTI of the median nerve at 3.0T. Optimal reconstruction parameters for fiber tractography should encompass a minimum FA threshold of 0.2 and a maximum angulation tolerance of 10.