Although mutation is commonly thought of as a random process, evolutionary studies show that different types of nucleotide substitution occur with widely varying rates that presumably reflect biases intrinsic to mutation and repair mechanisms. A strand asymmetry, the occurrence of particular substitution types at higher rates than their complementary types, that is associated with DNA replication has been found in bacteria and mitochondria. A strand asymmetry that is associated with transcription and attributable to higher rates of cytosine deamination on the coding strand has been observed in enterobacteria. Here, we describe a qualitatively different transcription-associated strand asymmetry in mammals, which may be a byproduct of transcription-coupled repair in germline cells. This mutational asymmetry has acted over long periods of time to produce a compositional asymmetry, an excess of G+T over A+C on the coding strand, in most genes. The mutational and compositional asymmetries can be used to detect the orientations and approximate extents of transcribed regions.