We present data on the frequencies of nucleotides and nucleotide substitutions in conservative DNA regions involved in the regulation of gene expression. Data on prokaryotes and eukaryotes are considered separately. In both cases DNA strands complementary to those which serve as templates for RNA-polymerase have low frequencies of cytosine. The most conservative positions also have an increased frequency of adenine. Various substitutions in the series of homologous regulatory DNA sequences, as compared to their consensuses, have different frequencies. In prokaryotes guanine in a consensus sequence is substituted for at the lowest and adenine at the highest frequency, whereas in eukaryotes cytosine is substituted for at the lowest and guanine at the highest frequency. In both cases the nucleotides substituted for are most frequently replaced with cytosine. Deviations from consensus sequences tend to cluster in adjacent positions. The more pronounced the consequences of a nucleotide substitution are the higher is the frequency of substitutions in adjacent positions. Possible explanations for these phenomena are discussed.