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Protein function shift can be predicted from sequence comparisons, either using positive selection signals or evolutionary rate estimation. None of the methods have been validated on large datasets, however. Here we investigate existing and novel methods for protein function shift prediction, and benchmark the accuracy against a large dataset of proteins with known enzymatic functions. Function change was predicted between subfamilies by identifying two kinds of sites in a multiple sequence alignment: Conservation-Shifting Sites (CSS), which are conserved in two subfamilies using two different amino acid types, and Rate-Shifting Sites (RSS), which have different evolutionary rates in two subfamilies. CSS were predicted by a new entropy-based method, and RSS using the Rate-Shift program. In principle, the more CSS and RSS between two subfamilies, the more likely a function shift between them. A test dataset was built by extracting subfamilies from Pfam with different EC numbers that belong to the same domain family. Subfamilies were generated automatically using a phylogenetic tree-based program, BETE. The dataset comprised 997 subfamily pairs with four or more members per subfamily. We observed a significant increase in CSS and RSS for subfamily comparisons with different EC numbers compared to cases with same EC numbers. The discrimination was better using RSS than CSS, and was more pronounced for larger families. Combining RSS and CSS by discriminant analysis improved classification accuracy to 71%. The method was applied to the Pfam database and the results are available at http://FunShift.cgb.ki.se. A closer examination of some superfamily comparisons showed that single EC numbers sometimes embody distinct functional classes. Hence, the measured accuracy of function shift is underestimated.