N-methylation at the arginine residues in RNA binding proteins with the arginine and glycine rich RGG box has been identified. We show that a synthetic peptide R9 (GGRGRGGGF) with the RGG sequence present in human fibrillarin and fragile X mental retardation protein (FMRP) can be specifically methylated by rat brain extract. A control peptide K9 with all arginines replaced by lysines could not be methylated under the same conditions, indicating that the arginines in the peptide were the methylation sites. A novel missense mutation, which changes an arginine to a histidine in the RGG box region of FMRP in a typical fragile X patient, has been identified. A synthetic peptide with this Arg-->His (GGRGHGGGF) substitution was methylated by our in vitro methylation system to a much less extent. Amino acid analysis of the methylated R9 peptide identified the methylated amino acid as monomethylarginine. The R9 peptide may be useful for further studies on arginine methylation in RGG proteins.