The kinetochore is a complex molecular machine that directs chromosome segregation during mitosis. It is one of the most elaborate subcellular protein structures in eukaryotes, comprising more than 100 different proteins. Inner kinetochore proteins associate with specialized centromeric chromatin containing the histone H3 variant centromere protein A (CENP-A) in place of H3. Outer kinetochore proteins bind to microtubules and signal to delay anaphase onset when microtubules are absent. Since the first kinetochore proteins were discovered and cloned 30 years ago using autoimmune sera from patients with scleroderma-spectrum disease, much has been learnt about the composition, functions and regulation of this remarkable structure.