A major obstacle to successful cytogenetic analysis of small intestinal crypt cells is the acquisition of a sufficient number of high-quality metaphases. A squash procedure has been developed for analysis of metaphase chromosomes from rat small intestine that largely circumvents this difficulty. The method involves a schedule of hypotonic treatment, fixation in ethanol: acetic acid, followed by maceration of the intestinal tissue in 3.5 N HCl. The procedure resulted in large numbers of well-spread, cytoplasm-free metaphases.