The non-protein amino acid homocysteine (Hcy) enters the first step of protein synthesis and forms an aminoacyl-tRNA synthetase-bound homocysteinyl adenylate (Hcy-AMP). Direct incorporation of Hcy into tRNA and protein is prevented by editing activities of aminoacyl-tRNA synthetases that convert Hcy-AMP into Hcy thiolactone. Editing of Hcy occurs in all cell types investigated, including human. S-Nitrosylation of Hcy prevents its editing by MetRS and allows formation of S-nitroso-Hcy-tRNAMet, as well as incorporation of Hcy into proteins at positions specified by methionine codons. This provides an example of how the genetic code can be expanded by invasion of the metionine coding pathway by Hcy. Hcy can also be incorporated into protein post-translationally by a facile reaction of Hcy thiolactone with ?-amino groups of protein lysine residues. Hcy is present in human blood proteins, such as hemoglobin, serum albumin, and ?-globulins. Hcy thiolactonase, a component of high-density lipoprotein, minimizes protein N-homocysteinylation. Incorporation of Hcy into protein provides plausible chemical mechanism by which elevated levels of Hcy contribute to human cardiovascular disease.