A detailed phylogenetic analysis for mammalian members of the family Herpesviridae, based on molecular sequences is reported. Sets of encoded amino acid sequences were collected for eight well conserved genes that are common to mammalian herpesviruses. Phylogenetic trees were inferred from alignments of these sequence sets using both maximum parsimony and distance methods, and evaluated by bootstrap analysis. In all cases the three recognised subfamilies (Alpha-, Beta- and Gammaherpesvirinae), and major sublineages in each subfamily, were clearly distinguished, but within sublineages some finer details of branching were incompletely resolved. Multiple-gene sets were assembled to give a broadly based tree. The root position of the tree was estimated by assuming a constant molecular clock and also by analysis of one herpesviral gene set (that encoding uracil-DNA glycosylase) using cellular homologues as outgroups. Both procedures placed the root between the Alphaherpesvirinae and the other two subfamilies. Substitution rates were calculated for the combined gene sets based on a previous estimate for alphaherpesviral UL27 genes, where the time base had been obtained according to the hypothesis of cospeciation of virus and host lineages. Assuming a constant molecular clock, it was then estimated that the three subfamilies arose approximately 180 to 220 million years ago, that major sublineages within subfamilies were probably generated before the mammalian radiation of 80 to 60 million years ago, and that speciations within sublineages took place in the last 80 million years, probably with a major component of cospeciation with host lineages.