The claudins comprise a multigene family of integral membrane proteins, which play a major role in tight junction formation. Aberrations in the expression of certain claudins have been described in a number of malignancies. Our aims were to determine the expression pattern of claudins 1, 3, and 4 as well as ZO-1 in a large series of US patients with gastric cancer and to correlate expression with clinicopathologic and prognostic variables. Tissue microarrays were created from paraffinized samples from 146 patients with distal gastric adenocarcinomas (61 intestinal and 85 diffuse or mixed subtypes). In addition, cores of normal mucosa and intestinal metaplasia were taken from most cases. The microarrays were stained for claudins 1, 3, and 4 and ZO-1, and the intensity of staining was determined using a 3-point scale. Moderate claudin 1 and ZO-1 membranous staining were present, whereas only focal weak claudin 3 and 4 membranous staining was present in normal gastric epithelium. Moderate to strong staining of claudins 1, 3, 4, and ZO-1 was detected in 74%, 48%, 62%, and 74% of the intestinal but in only 46%, 24%, 45%, and 36% of the diffuse subtype of adenocarcinomas (P < .05). Cox multivariate analysis revealed that tumor stage, diffuse subtype, and moderate to strong claudin 4 staining were associated with decreased survival (P < .02). In conclusion, claudins 1, 3, and 4 and ZO-1 are strongly expressed in most gastric intestinal-type adenocarcinomas but less frequently in diffuse gastric cancers. The up-regulation of claudin expression during gastric carcinogenesis suggests their potential utility as diagnostic biomarkers and possible targets for therapeutic intervention.