The specific recognition of genomic positive strand RNAS as templates for the synthesis of intermediate negative strands by the picornavirus replication machinery is presumably mediated by cis-acting sequences within the genomic RNA 3' non-coding region (NCR). A structure-infectivity analysis was conducted on the 44 nt human rhinovirus 14 (HRV14) 3' NCR to identify the primary sequence and/or secondary structure determinants required for viral replication. Using biochemical RNA secondary structure probing techniques, we have demonstrated the existence of a single stem-loop structure contained entirely within the 3' NCR, which appears to be phylogenetically conserved within the rhinovirus genus. We also report the in vivo analysis of a number of 3' NCR deletion mutations engineered into infectious cDNA clones which were designed to disrupt the stem-loop secondary structure to varying degrees. Large deletions (up to 37 nt) resulted in defective growth phenotypes, although they were not lethal. We propose that the absolute requirements for initiation of negative strand synthesis are less stringent than previously postulated, even though defined RNA secondary structure determinants may have evolved to facilitate and/or regulate the process of viral RNA replication.