Genetically encoded unnatural amino acids provide powerful strategies for modulating the molecular functions of proteins in mammalian cells. However, this approach has not been coupled to genome-wide measurements, because efficient incorporation of unnatural amino acids is limited to transient expression settings that lead to very heterogeneous expression. We demonstrate that stable integration of the Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)/tRNA(Pyl)CUA pair (and its derivatives) into the mammalian genome enables efficient, homogeneous incorporation of unnatural amino acids into target proteins in diverse mammalian cells, and we reveal the distinct transcriptional responses of embryonic stem cells and mouse embryonic fibroblasts to amber codon suppression. Genetically encoding N-ɛ-acetyl-lysine in place of six lysine residues in histone H3 enables deposition of pre-acetylated histones into cellular chromatin, via a pathway that is orthogonal to enzymatic modification. After synthetically encoding lysine-acetylation at natural modification sites, we determined the consequences of acetylation at specific amino acids in histones for gene expression.