The mammalian DYRK (dual specificity tyrosine phosphorylated and regulated kinase) family of protein kinases comprises a number of related, but poorly understood enzymes. DYRK1A is nuclear while DYRKs 2 and 3 are cytoplasmic. We recently showed that DYRK2 phosphorylates the translation initiation factor eIF2B at Ser539 in its epsilon-subunit and thereby "primes" its phosphorylation by glycogen synthase kinase-3. Here we have used peptides based on the sequence around Ser539 to help define the specificity of DYRK2/3 in comparison with DYRK1A. These kinases require an arginine N-terminal to the target residue for efficient substrate phosphorylation. This cannot be replaced even by lysine. A peptide with arginine at -2 is phosphorylated much less well by all three kinases than one with arginine at -3. Replacement of the +1 proline by alanine almost completely eliminates substrate phosphorylation, but valine here does allow phosphorylation especially by DYRK2. This study reveals both similarities and differences in the specificities of these arginine-dependent protein kinases.