Juvenile polyposis syndrome (JPS; OMIM 174900) is a rare disorder which is characterized by the presence of hamartomatous polyps throughout the gastrointestinal tract and an increased risk of gastrointestinal malignancy. Mutations of the SMAD4 gene on chromosome 18q21.1 have been shown to cause a subset of JPS cases, with estimates ranging from 20% to >50%. Characterization of the genes that cause the remainder of JPS cases relies on the certainty that SMAD4 is not the causative gene. We have undertaken a comprehensive analysis of germline SMAD4 mutations in a cohort of JPS patients to define the spectrum of mutations that cause JPS. We have analyzed a series of polyps from these patients for SMAD4 protein expression. We have also performed a blinded assessment of polyp material to look for morphological differences between polyps from patients with and without a germline SMAD4 mutation. The results indicate that almost all germline SMAD4 mutations are readily detectable by screening genomic DNA using polymerase chain reaction-based methods; SMAD4 can be excluded as the causative gene in the majority of our JPS cohort. Loss of SMAD4 expression occurs in most polyps from SMAD4 mutation carriers, even those with missense germline mutations. SMAD4 loss in polyps is, however, not a feature of cases that are not caused by SMAD4 mutations, indicating that these polyps develop along a SMAD4-independent pathway. The morphology of polyps from SMAD4 mutation carriers is subtly different from other JPS polyps, notably including a more prominent epithelial component in the former.