The halophilic archaebacterium, Haloarcula marismortui, contains two nonadjacent ribosomal RNA operons, designated rrnA and rrnB, in its genome. The 16S rRNA genes within these operons are 1472 nucleotides in length and differ by nucleotide substitutions at 74 positions. The substitutions are not uniformly distributed but rather are localized within three domains of 16S rRNA; more than two-thirds of the differences occur within the domain bounded by nucleotides 508 and 823. This domain is known to be important for P site binding of aminoacylated tRNA and for 30-50S subunit association. Using S1 nuclease protection, it has been shown that the 16S rRNAs transcribed from both operons are equally represented in the functional 70S ribosome population. Comparison of these two H. marismortui sequences to the 16S gene sequences from related halophilic genera suggests that (i) in diverging genera, mutational differences in 16S gene sequences are not clustered but rather are more generally distributed throughout the length of the 16S sequence, and (ii) the rrnB sequence, particularly within the 508-823 domain, is more different from the out group sequences than is the rrnA sequence. Several possible explanations for the evolutionary origin and maintenance of this sequence heterogeneity within 16S rRNA of H. marismortui are discussed.