Autoinhibitory domains are regions of proteins that negatively regulate thefunction of other domains via intramolecular interactions. Autoinhibition is apotent regulatory mechanism that provides tight “on-site”repression. The discovery of autoinhibition generates valuable clues to how aprotein is regulated within a biological context. Mechanisms that counteractthe autoinhibition, including proteolysis, post-translational modifications, aswell as addition of proteins or small molecules intrans, oftenrepresent central regulatory pathways. In this review, we document thediversity of instances in which autoinhibition acts in cell regulation. Sevenwell-characterized examples (e.g., σ⁷⁰, Ets-1, ERM, SNARE andWASP proteins, SREBP, Src) are covered in detail. Over thirty additionalexamples are listed. We present experimental approaches to characterizeautoinhibitory domains and discuss the implications of this widespreadphenomenon for biological regulation in both the normal and diseasedstates.