Editing of the glutamate receptor subunit B (GluR-B) pre-mRNA at a single adenosine residue results in an amino-acid change that profoundly alters the electrophysiological properties of the receptor. Here we show that the GluR-B pre-mRNA is efficiently and accurately edited in vitro, and that base-pair interactions between the editing site and a sequence in the downstream intron are required for substrate recognition. In addition, we directly demonstrate that editing results from the conversion of adenosine to inosine by enzymatic deamination. The biochemical properties of this GluR-B editing activity are similar to those of a double-stranded-RNA-dependent adenosine deaminase, but RNA competition and column fractionation experiments indicate that the GluR-B editing and deaminase activities are distinct. Thus, the GluR-B editing enzyme may contain the adenosine deaminase, or a similar activity, and an RNA recognition subunit that specifically targets the enzyme to the editing site.