The current convention in estimating the number of substitutions per synonymous site (K
) and per nonsynonymous site (K
) between two protein-coding genes is to count each twofold degenerate site as one-third synonymous and two-thirds nonsynonymous because one of the three possible changes at such a site is synonymous and the other two are nonsynonymous. This counting rule can considerably overestimate theK
value because transitional mutations tend to occur more often than transversional mutations and because most transitional mutations at twofold degenerate sites are synonymous. A new method that gives unbiased estimates is proposed. An application of the new and the old method to 14 pairs of mouse and rat genes shows that the new method gives aK
value very close to the number of substitutions per fourfold degenerate site whereas the old method gives a value 30% higher. Both methods give aK
value close to the number of substitutions per nondegenerate site.