In this study, we show that it is possible to increase the performance over PSI-BLAST by using evolutionary information for both query and target sequences. This information can be used in three different ways: by sequence linking, profile-profile alignments, and by combining sequence-profile and profile-sequence searches. If only PSI-BLAST is used, 16% of superfamily-related protein domains can be detected at 90% specificity, but if a sequence-profile and a profile-sequence search are combined, this is increased to 20%, profile-profile searches detects 19%, whereas a linking procedure identifies 22% of these proteins. All three methods show equal performance, but the best combination of speed and accuracy seems to be obtained by the combined searches, because this method shows a good performance even at high specificity and the lowest computational cost. In addition, we show that the E-values reported by all these methods, including PSI-BLAST, underestimate the true rate of false positives. This behavior is seen even if a very strict E-value cutoff and a limited number of iterations are used. However, the difference is more pronounced with a looser E-value cutoff and more iterations. Copyright 2003 Wiley-Liss, Inc.