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Hi Matt, You're right, the images are aligned to each other. What I meant to write was that, for some subjects, the output matrix of the diffusion/structural/freesurfer registration (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FDT/UserGuide#FreeSurfer_Registration) was calculated incorrectly. Since I pass this matrix (freesurfer2fa.mat) into the probtrackx2 command, the analysis would be incorrect if the transform is off. In the past, I have found that skull-stripping can help in instances of problematic registration. For my previously problematic HCP subjects, I found that when I added a skull-stripping step before computing this matrix, the freesurfer2fa.mat was now computed correctly. Thanks, Jonathan