Intestinal epithelial cells derive from stem cells at the base of the crypt and migrate along the crypt-lumen axis. Their life is terminated as they reach the luminal surface where they detach and are shed. Intestinal epithelial cells show evidence of apoptosis in the region of shedding, and cell death is thought to resemble a form of apoptosis called detachment-induced cell death, or anoikis. Human intestinal epithelial cells die rapidly in vitro due to loss of anchorage during isolation, making primary culture of these cells a goal that has not yet been reached. However, the molecular mechanisms underlying this process of anoikis are largely unknown. In this study, a novel protocol for the rapid, temperature-controlled isolation of highly purified human colonic epithelial cells from surgical specimens is described. Using this method, early molecular events of anoikis in nontransformed epithelial cells were studied. Intestinal epithelial cells were isolated at the beginning of the apoptotic cascade, before the activation of caspase 3 family members and cleavage of poly(ADP-ribose) polymerase and DNA fragmentation. Elucidating the molecular mechanisms of detachment-induced cell death may facilitate the establishment of long-term primary cultures of human intestinal epithelial cells and enhance our understanding of homeostasis in the intestinal epithelium.