The p53-regulated gene product p21^WAF1/CIP1 is the prototype of a family of small proteins that negatively regulate the cell cycle. To learn more about p21^WAF1/CIP1 regulation in vivo, monoclonal antibodies were developed for immunohistochemistry. These revealed that p21^WAF1/CIP1 expression followed radiation-induced DNA damage in human skin in a pattern consistent with its regulation by p53. A detailed comparison of the human, rat, and mouse p21^WAF1/CIP1 promoter sequences revealed that this induction was probably mediated by conserved p53-binding sites upstream of the transcription start site. In unirradiated tissues, p21^WAF1/CIP1 expression was apparently independent of p53 and was observed in a variety of cell types. Moreover, there was a striking compartmentalization of p21^WAF1/CIP1 expression throughout the gastrointestinal tract that correlated with proliferation rather than differentiation. As epithelial cells migrated up the crypts, the K167-expressing proliferating compartment near the crypt base ended abruptly, with the coincident appearance of a nonproliferating compartment expressing p21^WAF1/CIP1. In colonic neoplasms, this distinct compartmentalization was largely abrogated. Cell cycle inhibitors are thus subject to precise topological control, and escape from this regulation may be a critical feature of neoplastic transformation.