It has been reported previously that the Human rhinovirus 14 (HRV-14) RNA genome contains a cis-acting replication element (cre) that maps to the capsid coding (P1) sequence [19]. Further characterization of the HRV-14 cre in the present study established that by moving the cre stem-loop structure downstream, adjacent to the 3'NCR, that its position is not critical for function. When the P1 sequences of two closely related serotypes of HRV-14 were analyzed for the presence of a cre, both HRV-3 and HRV-72 were found to contain similar sequence at the same positions as HRV-14. Moreover, sequence at these positions produced structures from MFOLD analysis that closely resembled the HRV-14 cre. It was also discovered that neither HRV serotypes 1a or 16 harbor replication elements that map to the P1 segments of their genomes. Computer and mutational analyses suggest that the cre in these latter HRV serotypes map instead to the 2A gene, as has been reported for HRV-2. The putative HRV-3 cre was determined to be unable to support replication when placed in an HRV-14 replicon background. Similarly, the previously identified HRV-2 cre was unable to support replication of the HRV-14 genome. This finding is in contrast to the cardiovirus cre, which has been shown to be functionally active between two members of its family, and further suggests that there is a close link between the evolution of the human rhinoviruses and the mechanisms of RNA replication.