We have calibrated five different molecular clocks for circulating poliovirus based upon the rates of fixation of total substitutions (K t ), synonymous substitutions (K s ), synonymous transitions (A s ), synonymous transversions (B s ), and nonsynonymous substitutions (K a ) into the P1/capsid region (2,643 nucleotides). Rates were determined over a 10-year period by analysis of sequences of 31 wild poliovirus type 1 isolates representing a well-defined phylogeny derived from a common imported ancestor. Similar rates were obtained by linear regression, the maximum likelihood/single-rate dated-tip method, and Bayesian inference. . Nonsynonymous substitutions at all P1/capsid sites, including the neutralizing antigenic sites, appeared to be constrained by purifying selection. Simulation of the evolution of third-codon positions suggested that saturation of synonymous transitions would be evident at 10 years and complete at ϳ65 years of independent transmission. Saturation of synonymous transversions was predicted to be minimal at 20 years and incomplete at 100 years. The rapid evolution of the K t , K s , and A s clocks can be used to estimate the dates of divergence of closely related viruses, whereas the slower B s and K a clocks may be used to explore deeper evolutionary relationships within and across poliovirus genotypes.Poliovirus is one of the most rapidly evolving viruses known (17,31,38,52,58). Estimates of the rates of total nucleotide substitution into poliovirus capsid regions average ϳ10 Ϫ2 substitutions per site per year (31, 39, 52-54, 89, 90). The rates appear to be similar across the three poliovirus serotypes and for both circulating polioviruses and polioviruses associated with chronic infections. This very rapid rate of genomic evolution has facilitated high-resolution molecular epidemiologic studies, permitting the unambiguous identification of the sources of imported viruses (10, 41) and the resolution of separate lineages during outbreaks (39,43,74,75), during endemic transmission (23,52,90), during prolonged poliovirus replication in immunodeficient patients (9,31,53,89), and from environmental surveillance (23). However, the rapid accumulation of nucleotide substitutions, most of which are synonymous transitions, obscures deeper evolutionary relationships (46,70).Underlying the rapid pace of poliovirus genomic evolution are the high rates of base misincorporation (in the range of 10 Ϫ5 to 10 Ϫ3 per base per replication) by the poliovirus RNAdependent RNA polymerase (2,15,19,81,82,84). These high mutation rates are attributable to the absence of 3Ј35Ј exonuclease proofreading mechanisms for the viral RNA polymerases (19), although other mechanisms may also be involved (17, 18). The strong transition bias of the poliovirus polymerase (2, 46) is reflected in the pattern of fixation nucleotide substitutions into poliovirus genomes.In this report, we have calibrated five different molecular clocks based upon the rates of fixation of total substitutions (K t ), synonymous substitutions (K s ),...