Pseudomembranous colitis is often caused by Clostridium difficile; however, it may also be due to ischemia. To determine if any histologic features could be used to differentiate C difficile from ischemia, 49 biopsies of pseudomembranous colitis (25 from patients with C difficile colitis and 24 from patients with ischemic colitis) were coded, randomized, and evaluated for the presence of numerous variables, including the amount and distribution of mucosal necrosis, lamina propria hyalinization, and atrophic "micro-crypts." Hyalinization of the lamina propria was seen in 19 cases of ischemia but not in C difficile colitis (p < 0.0001). Atrophic-appearing micro-crypts were seen in 18 ischemic cases and 6 C difficile cases (p < 0.0006). Lamina propria hemorrhage, full-thickness mucosal necrosis, and a diffuse microscopic distribution of pseudomembranes were significantly more common in ischemia than C difficile. Endoscopic examination identified pseudomembranes significantly more often with C difficile than ischemia, while the endoscopic appearance of masses or polyps was seen exclusively in cases of ischemia. The presence of a hyalinized lamina propria appeared to be a specific and sensitive marker for ischemia in colon biopsies with pseudomembranes. The presence of atrophic micro-crypts, lamina propria hemorrhage, full-thickness mucosal necrosis, diffuse involvement of all the surface of all biopsies by pseudomembranes, and the endoscopic impression of a localized process, polyp, or mass were also markers of ischemia, while the endoscopic identification of diffuse pseudomembranes favored the diagnosis of C difficile.