Gene duplication provides the opportunity for subsequent refinement of dist
inct functions of the duplicated copies. Either through changes in coding s
equence or changes in regulatory regions, duplicate copies appear to obtain
new or tissue-specific functions. If this divergence were driven by natura
l selection, we would expect duplicated copies to have differentiated patte
rns of substitutions. We tested this hypothesis using genes that duplicated
before the human/mouse split and whose orthologous relations were clear. T
he null hypothesis is that the number of amino acid changes between humans
and mice was distributed similarly across different paralogs. We used a met
hod modified from Tang and Lewontin to detect heterogeneity in the amino ac
id substitution pattern between those different paralogs. Our results show
that many of the paralogous gene pairs appear to be under differential sele
ction in the human/mouse comparison. The properties that led to diversifica
tion appear to have arisen before the split of the human and mouse lineages
. Further study of the diverged genes revealed insights regarding the patte
rns of amino acid substitution that resulted in differences in function and
/or expression of these genes. This approach has utility in the study of ne
wly identified members of gene families in genomewide data mining and for c
ontrasting the merits of alternative hypotheses for the evolutionary diverg
ence of function of duplicated genes.