This study is concerned with the isolation and characterization of the enzyme, S-adenosylmethionine:ribosomal ribonucleic acid-adenine (N(6-)) methyl-transferase [rRNA-adenine (N(6)-) methylase] of Escherichia coli strain B, which is responsible for the formation of N(6)-methyladenine moieties in ribosomal ribonucleic acids (rRNA). A 1,500-fold purified preparation of the species-specific methyltransferase methylates a limited number of adenine moieties in heterologous rRNA (Micrococcus lysodeikticus and Bacillus subtilis) and methyl-deficient homologous rRNA. The site recognition mechanism does not require intact 16 or 23S rRNA. The enzyme does not utilize transfer ribonucleic acid as a methyl acceptor nor does it synthesize 2-methyladenine or N(6)-dimethyladenine moieties. Mg(2+), spermine, K(+), and Na(+) increase the reaction rate but not the extent of methylation; elevated concentrations of the cations inhibit markedly. The purified preparations utilize 9-beta-ribosyl-2,6-diaminopurine (DAPR) as a methyl acceptor with the synthesis of 9-beta-ribosyl-6-amino-2-methylaminopurine. A comparison of the two activities demonstrated that one methyltransferase is responsible for the methylation of both DAPR and rRNA. This property provides a sensitive assay procedure unaffected by ribonucleases and independent of any specificity exhibited by rRNA methyl acceptors.