Background: Several entropy-based methods have been developed for scoring sequence conservation in protein multiple sequence alignments. High scoring amino acid positions may correlate with structurally or functionally important residues. However, amino acid background frequencies are usually not taken into account in these entropy-based scoring schemes. Results: We demonstrate that using a relative entropy measure that incorporates amino acid background frequency results in improved performance in identifying functional sites from protein multiple sequence alignments. Conclusion: Our results suggest that the application of appropriate background frequency information may lead to more biologically relevant results in many areas of bioinformatics. Background Protein multiple sequence alignments are widely used to infer conservation of amino acid residues within an evolutionarily related family [1,2]. Highly conserved residues tend to correlate with structural and/or functional importan...