The cis-acting sequence elements that direct measles virus (MV) genome synthesis reside in the 109 base non-coding region at the 5' trailer (3' antigenomic) end of MV genome that makes up the antigenomic promoter (AGP). The MV-AGP nucleotides 79-96, corresponding to nucleotide hexamers 14, 15 and 16 (the C' element), show sequence similarity with the equivalent region of many paramyxoviruses and are analogous to the three nucleotide hexamers that form the second replication control element in the Sendai virus AGP. In this study, results of two independent procedures demonstrate that the MV C' element also is a replication control sequence. Results of in vivo nucleotide selection experiments show that selection pressure for retaining the wild type nucleotides at the first position of each of the three hexamers, and for the fifth position of the 14th hexamer was relatively high. However, with continued replication, preference for the conservation of wild type nucleotides across the entire C' element was clearly evident. Results of mutational analysis of individual nucleotides in one or more hexamers in a measles-helper-virus driven reporter gene rescue system agreed with these results. Substitutions at the first position of the 14th, the 15th or the 16th hexamers reduced minireplicon activity dramatically. In contrast, changes at the other five positions of any one hexamer had little or no effect on minireplicon activity, even when all the five bases were changed at the same time. However, when minireplicons were analyzed which contained point mutations at equivalent positions in all three hexamers, it was evident that the nucleotides, particularly those at the 5th position, were also important components of the C' element. This pattern of sequence requirement in the C' element based on mutational analysis could be described as a distinct motif, 5'-(GNNNAN)2GNNNCN-3', that is important for MV replication.