The isochore theory depicts the genomes of warm-blooded vertebrates as a mosaic 
of long genomic regions that are characterized by relatively homogeneous GC 
content. In the absence of genomic data, the GC content at third-codon positions 
of protein-coding genes (GC3) was commonly used as a proxy for the GC content of 
isochores. Oddly, in the postgenomic era, GC3 is still sometimes used as a proxy 
for the GC composition of isochores. Here, we use genic and genomic sequences 
from human, chimpanzee, cow, mouse, rat, chicken, and zebrafish to show that GC3 
only explains a very small proportion of the variation in GC content of long 
genomic sequences flanking the genes (GCf), and what little correlation there is 
between GC3 and GCf was found to decay rapidly with distance from the gene. The 
coefficient of variation of GC3 was found to be much larger than that of GCf 
and, therefore, GC3 and GCf values are not comparable with each other. 
Comparisons of orthologous gene pairs from 1) human and chimpanzee and 2) mouse 
and rat show strong correlations between their GC3 values, but very weak 
correlations between their GCf values. We conclude that the GC content of 
third-codon position cannot be used as stand-in for isochoric composition.