The standard genetic test for Lynch syndrome (LS) frequently reveals an absence of pathogenic mutations in DNA mismatch repair genes known to be associated with LS. It was recently shown that germ line deletions in the last exons of EPCAM are involved in the etiology of LS. The aim of this study was to evaluate the prevalence of EPCAM deletions in a Spanish population and the clinical implications of deletion. Probands from 501 families suspected of having LS were enrolled in the study. Twenty-five cases with MSH2 loss were identified: 10 had mutations of MSH2, five had mutations of MSH6, and 10 did not show MSH2/MSH6 mutations. These 25 cases were analyzed for EPCAM deletions using multiplex ligation-dependent probe amplification, and deletions were mapped using long-range PCR analysis. One subject with no MSH2/MSH6 mutations had a large deletion in the EPCAM locus that extended for 8.7 kb and included exons 8 and 9. The tumor exhibited MSH2 promoter hypermethylation. EPCAM deletion analysis followed by MSH2 methylation testing of the tumor is a fast low-cost procedure that can be used to identify mutations that cause LS. We propose that this procedure be incorporated into clinical genetic analysis strategies and present a decision-support flow diagram for the diagnosis of LS. Copyright © American Society for Investigative Pathology and the Association for Molecular Pathology.