Abstract Motivation: An automatic sequence searching method (ProtEST) is described which constructs multiple protein sequence alignments from protein sequences and translated expressed sequence tags (ESTs). ProtEST is more effective than a simple TBLASTN search of the query against the EST database, as the sequences are automatically clustered, assembled, made non-redundant, checked for sequence errors, translated into protein and then aligned and displayed. Results: A ProtEST search found a non-redundant, translated, error- and length-corrected EST sequence for &58% of sequences when single sequences from 1407 Pfam-A seed alignments were used as the probe. The average family size of the resulting alignments of translated EST sequences contained &10 sequences. In a cross-validated test of protein secondary structure prediction, alignments from the new procedure led to an improvement of 3.4% average Q3 prediction accuracy over single sequences. Availability: The ProtEST method is available as an Internet World Wide Web service at http://barton.ebi.ac.uk/servers/protest.htmlThe Wise2 package for protein and genomic comparisons and the ProtESTWise script can be found at: http://www.sanger.ac.uk/Software/Wise2 Contact: geoff@ebi.ac.uk