The genomes of mammals and birds can be partitioned into megabase-long regions, termed isochores, with consistently high, or low, average C + G content. Isochores with high CG contain a mixture of CG-rich and AT-rich genes, while high-AT isochores contain predominantly AT-rich genes. The two gene populations in the high-CG isochores are functionally distinguishable by statistical analysis of their gene ontology categories. However, the aggregate of the two populations in CG isochores is not statistically distinct from AT-rich genes in AT isochores. Genes tend to be located at local extrema of composition within the isochores, indicating that the CG-enriching mechanism acted differently when near to genes. On the other hand, maximum-likelihood reconstruction of molecular phylogenetic trees shows that branch lengths (evolutionary distances) for third codon positions in CG-rich genes are not substantially larger than those for AT-rich genes. In the context of neutral mutation theory this argues against any strong positive selection. Disparate features of isochores might be explained by a model in which about half of all genes functionally require AT richness, while, in warm-blooded organisms, about half the genome (in large coherent blocks) acquired a strong bias for mutations to CG. Using mutations in CG-rich genes as convenient indicators, we show that approximately 20% of amino acids in proteins are broadly substitutable, without regard to chemical similarity.