Previous studies have identified three brain proteins (40, 65 and 95 kDa, respectively) that specifically bind to the 3'-untranslated region of GAP-43 mRNA. In this study, using a specific monoclonal antibody, we now show that the 40-kDa proteins are members of the Elav-like protein family. This family of specific RNA-binding proteins comprise three neural specific members called HuD, HuC, and Hel-N1. We have shown that purified recombinant HuD can bind with high affinity to GAP-43 mRNA. In addition, we have mapped the binding site to a highly conserved 26-nucleotide sequence within the regulatory element. The binding of HuD to this site is readily displaced by RNA oligonucleotides encoding other HuD binding sites. We also show that only the first and second RNA binding domains of HuD are required for selective binding to GAP-43 mRNA.