The X-ray crystal structure of the tyrosine kinase domain of the human insulin receptor has been determined by multiwavelength anomalous diffraction phasing and refined to 2.1 Angstrom resolution. The structure reveals the determinants of substrate preference for tyrosine rather than serine or threonine and a novel autoinhibition mechanism whereby one of the tyrosines that is autophosphorylated in response to insulin, Tyr 1,162, is bound in the active site.