The impact of the somatic hypermutational machinery was examined by analyzing the frequency and distribution of mutations in nonproductive V(H)DJ(H) rearrangements obtained from individual human peripheral B cells. A strong bias toward nucleotide substitutions within the quadruplet motif RGYW was observed. In addition, there was a comparably increased frequency of mutations of the inverse repeat of RGYW, WRCY. Together, mutations of RGYW/WRCY accounted for 37% of all nucleotide substitutions. No significant strand polarity of the distribution of mutations was evident when nucleotide substitutions of highly mutated quartets and triplets as well as of their inverse repeats were analyzed. Furthermore, detailed analysis of mutations of specific triplets, such as AGC and TAC provided evidence that they were mutated more frequently when they were included within RGYW and WRCY, respectively. Despite being a target of the mutational machinery, neither RGYW nor WRCY was mutated in the absence of a large number of substitutions of other nucleotides in the same sequence. These results indicate that the mutational machinery targets RGYW sequences for mutations on either DNA strand and do not support the contention that the mutational machinery exhibits DNA strand polarity.