Human cDNA clones encoding the UUAG-binding heterogeneous nuclear ribonucleoprotein (hnRNP) D0 protein have been isolated and expressed. The protein has two RNA-binding domains (RBDs) in the middle part of the protein and an RGG box, a region rich in glycine and arginine residues, in the C-terminal part ("2xRBD-Gly" structure). The hnRNP A1, A2/B1, and D0 proteins, all possess common features of the 2xRBD-Gly structure and binding specificity toward RNA. Together, they form a subfamily of RBD class RNA binding proteins (the 2xRBD-Gly family). One of the structural characteristics shared by these proteins is the presence of several isoforms presumably resulting from alternative splicing. Filter binding assays, using the recombinant hnRNP D0 proteins that have one of the two RBDs, indicated that one RBD specifically binds to the UUAG sequence. However, two isoforms with or without a 19-amino acid insertion at the N-terminal RBD showed different preference toward mutant RNA substrates. The 19-amino acid insertion is located in the N-terminal end of the first RBD. This result establishes the participation of the N terminus of RBD in determining the sequence specificity of binding. A similar insertion was also reported with the hnRNP A2/B1 proteins. Thus, it might be possible that this type of insertion with the 2xRBD-Gly type RNA binding proteins plays a role in "fine tuning" the specificity of RNA binding. RBD is supposed to bind with RNA in general and sequence-specific manners. These two discernible binding modes are proposed to be performed by different regions of the RBD. A structural model of these two binding sites is presented.