The amino acid substitutions at a site are affected by mixture of various constraints. It is also known that the amino acid substitutions are accelerated at sites under positive selection. However, the relationship between the substitutions at positively selected sites and the constraints has not been thoroughly examined. The advances in computational biology have enabled us to divide the mixture of the constraints into the structural constraint and the remainings by using the amino acid sequences and the tertiary structures, which is expressed as the deviation of the mixture of constraints from the structural constraint. Here, two types of profiles, or matrices with the size of 20 x (site length), are compared. One of the profiles represents the mixture of constraints, and is generated from a multiple amino acid sequence alignment, whereas the other is designed to represent the structural constraints. We applied the profile comparison method to proteins under positive selection to examine the relationship between the positive selection and constraints. The results suggested that the constraint at a site under positive selection tends to be deviated from the structural constraint at the site. (C) 2016 Elsevier Ireland Ltd. All rights reserved.