We present a new approach for analyzing directional mutation pressure and 
nucleotide content in protein-coding genes. Directional mutation pressure, the 
heterogenicity in the likelihood of different nucleotide substitutions, is used 
to explain the increasing or decreasing guanine-cytosine content (GC%) in DNA 
and is represented by microD, in agreement with Sueoka (1962, Proc Natl Acad Sci 
USA 48:582-592). The new method uses simulation to facilitate identification of 
significant A+T or G+C pressure as well as the comparison of directional 
mutation pressure among genes, even when they are translated by different 
genetic codes. We use the method to analyze the evolution of directional 
mutation pressure and nucleotide content of mitochondrial cytochrome b genes. 
Results from a survey of 110 taxa indicate that the cytochrome b genes of most 
taxa are subjected to significant directional mutation pressure and that the 
gene is subject to A+T pressure in most cases. Only in the anseriform bird 
Cairina moschata is the cytochrome b gene subject to significant G+C pressure. 
The GC% at nonsynonymous codon sites decreases proportionately with increasing 
A+T pressure, and with a slope less than one, indicating a presence of selective 
constraints. The cytochrome b genes of insects, nematodes, and eumycotes are 
subject to extreme A+T pressures (microD = 0.123, 0.224, and 0.130) and, in 
parallel, the GC% of the nonsynonymous codon sites has decreased from about 0.44 
in organisms that are not subjected to A+T or G+C pressure to about 0.332, 
0.323, and 0.367, respectively. The distribution of taxa according to the GC% at 
nonsynonymous codon sites and directional mutation pressure supports the notion 
that variation in these parameters is a phylogenetic component.