It has been claimed that synonymous sites in mammals are under selective 
constraint. Furthermore, in many studies the selective constraint at such sites 
in primates was claimed to be more stringent than that in rodents. Given the 
larger effective population sizes in rodents than in primates, the theoretical 
expectation is that selection in rodents would be more effective than that in 
primates. To resolve this contradiction between expectations and observations, 
we used processed pseudogenes as a model for strict neutral evolution, and 
estimated selective constraint on synonymous sites using the rate of 
substitution at pseudosynonymous and pseudononsynonymous sites in pseudogenes as 
the neutral expectation. After controlling for the effects of GC content, our 
results were similar to those from previous studies, i.e., synonymous sites in 
primates exhibited evidence for higher selective constraint that those in 
rodents. Specifically, our results indicated that in primates up to 24% of 
synonymous sites could be under purifying selection, while in rodents synonymous 
sites evolved neutrally. To further control for shifts in GC content, we 
estimated selective constraint at fourfold degenerate sites using a maximum 
parsimony approach. This allowed us to estimate selective constraint using 
mutational patterns that cause a shift in GC content (GT ↔ TG, CT ↔ TC, GA ↔ AG, 
and CA ↔ AC) and ones that do not (AT ↔ TA and CG ↔ GC). Using this approach, we 
found that synonymous sites evolve neutrally in both primates and rodents. 
Apparent deviations from neutrality were caused by a higher rate of C → A and C 
→ T mutations in pseudogenes. Such differences are most likely caused by the 
shift in GC content experienced by pseudogenes. We conclude that previous 
estimates according to which 20-40% of synonymous sites in primates were under 
selective constraint were most likely artifacts of the biased pattern of 
mutation.