Functional constraints on proteins limit their evolutionary rates at specific sites. These constraints allow for the interpretation of conserved residues and sites with a rate change as those most likely underlying the functional similarities and differences among protein subfamilies, respectively. This study describes new likelihood-ratio tests (LRTs) that complement existing ones for the identification of both conserved and rate change sites. These identifications are validated by the recovery of residues that are known from existing biochemical and structural information to be critical for the functional similarities and differences among carbonic anhydrases (CAs). In combination with this other information, these LRTs also support a unique antioxidant defense role for the puzzling CA III. As illustrated by the CAs, these LRTs, in combination with other biological evidence, offer a powerful and cost-effective approach for testing hypotheses, making predictions, and designing experiments in protein functional studies.