The N‐end rule‐based degradation signal, which targets a protein for ubiquitin‐dependent proteolysis, comprises a destabilizing amino‐terminal residue and a specific internal lysine residue. We report the isolation and functional analysis of a gene (UBR1) for the N‐end recognizing protein of the yeast Saccharomyces cerevisiae. UBR1 encodes a approximately 225 kd protein with no significant sequence similarities to other known proteins. Null ubr1 mutants are viable but are unable to degrade the substrates of the N‐end rule pathway. These mutants are partially defective in sporulation and grow slightly more slowly than their wild‐type counterparts. The UBR1 protein specifically binds in vitro to proteins bearing amino‐terminal residues that are destabilizing according to the N‐end rule, but does not bind to otherwise identical proteins bearing stabilizing amino‐terminal residues.