Noninvasive mapping of white matter tracts using diffusion tensor magnetic resonance imaging (DTMRI) is potentially useful in revealing anatomical connectivity in the human brain. However, a gold standard for validating DTMRI in defining axonal fiber orientation is still lacking. This study presents the first validation of the principal eigenvector of the diffusion tensor in defining axonal fiber orientation by superimposing DTMRI with manganese-enhanced MRI of optic tracts. A rat model was developed in which optic tracts were enhanced by manganese ions. Manganese ion (Mn2+) is a potent T1-shortening agent and can be uptaken and transported actively along the axon. Based on this property, we obtained enhanced optic tracts with a T1-weighted spin-echo sequence 10 h after intravitreal injection of Mn2+. The images were compared with DTMRI acquired with exact spatial registration. Deviation angles between tangential vectors of the enhanced tracts and the principal eigenvectors of the diffusion tensor were then computed pixel by pixel. We found that under signal-to-noise (SNR) of 30, the variance of deviation angles was (13.27°)2. In addition, the dependence of this variance on SNR obeys stochastic behavior if SNR is greater than 10. Based on this relation, we estimated that an rms deviation of less than 10° could be achieved with DTMRI when SNR is 40 or greater. In conclusion, our method bypasses technical difficulties in conventional histological approach and provides an in vivo gold standard for validating DTMRI in mapping white matter tracts.