The aim of this study was to investigate the biochemical basis of biomechanical and morphological alterations of upper esophageal sphincter, which have been reported in patients with Zenker's diverticulum. 4-L-Hydroxyproline (4-Hyp) (collagen), isodesmosine (Ides), and desmosine (Des) (elastin) contents were measured in samples of cricopharyngeal muscle (CPM) and muscularis propria of the esophagus below the CPM. The specimens were collected from seven patients operated for Zenker's diverticulum and eight cadavers, without esophageal and connective tissue disease, 4-Hyp was assayed colorimetrically, Ides and Des by high-performance liquid chromatography. Mean (+/-SEM) values were compared by Mann-Whitney U test. In patients, collagen content was significantly increased, both in CPM and in the muscularis propria of the esophagus below the CPM (P < 0.05). In CPM, Ides to Des and collagen to elastin ratios were significantly higher in patients than in controls (P < 0.05). Both the CPM and the upper muscular cuff of the esophagus appear to be involved in the pathogenesis of Zenker's diverticulum. This finding supports the extension of the myotomy to the muscularis propria of the esophagus below the CPM. The alterated Ides to Des ratio suggests a primary disease of CPM as a cause of Zenker's diverticulum.