This study describes sequence variation in both the enhancer and promoter segments of the upstream regulatory region (URR) of 28 human papillomavirus (HPV) type 18 isolates from cervical cancers, 25 from women treated at an Australian center and three from overseas included for comparison. No large-scale changes were found in either region. Fourteen substitutions were identified in the enhancer region with the number in individual isolates ranging from one to eight. Four substitutions impacted cellular transcription factor binding sites but there were no obvious associations with clinicopathological variables. The promoter segment was found to be more highly conserved than the enhancer, but four of the five point mutations identified involved cellular transcription factor binding motifs including a substitution of C for T at nt 104 which affected 21 samples. This change was found to impact upon a previously unrecognized Yin Yang (YY1) binding site. Electrophoretic mobility shift assays (EMSA) showed that this substitution significantly reduced protein-DNA binding and evidence was sought for its possible clinical implications. Of the 24 women with less than Stage III disease and known clinical outcome, tumor recurrence was observed in all of the 6 women whose isolates had the "prototype" T at nt 104, whereas only 8 of the 18 cancers with the mutation at this YY1 site recurred. This is the first report on URR variation in HPV 18 isolates from the South Pacific region. The study also provides initial data on diversity in the promoter region and preliminary evidence suggesting that a specific point mutation in this region may be clinically significant.