Purpose: To determine the total direct costs (fixed and variable costs) of diffusion tensor imaging (DTI) and MR tractography reconstruction of the brain. Materials and Methods: The direct fixed and variable costs of DTI with MR tractography were determined prospectively with time and motion analysis in a 1.5-Tesla MR scanner using 15 encoding directions. Seventeen patients with seizure disorders, 9 males & 8 females, with mean age of 13 years (age range 2 - 33 years) were studied. Total direct costs were calculated from all direct fixed and variable costs. Sensitivity analyses between 1.5 versus a 3-Tesla MR system, and 15 versus 32 encoding directions were done. Results: The total direct costs of DTI and MR tractography for a 1.5-T system with 15 encoding directions were US $97. Variable cost was $76.80 and fixed cost was $20.20. Total direct costs for a 3-T system with 15 directions decreased to US $94.5 because of the shorter scan time despite the higher cost of the 3-T system. The most costly component of the direct cost was post-processing analysis at US $46.00. Conclusion: DTI with MR tractography has important total direct costs with variable costs higher than the fixed costs. The post processing variable cost is the most expensive component. Developing more accurate automated post-processing software for DTI and MR tractography is important to decrease this variable labor cost. Given the added value of DTI-MR tractography and the costs involved reimbursement codes should be considered.