NP220 is a DNA-binding nuclear protein originally cloned from human cell lines. Human NP220 (hNP220) has an arginine/serine-rich motif found in non-small nuclear RNP splicing factors (SR proteins) and shares three domains (MH1, MH2 and MH3) with an acidic nuclear matrix protein, matrin 3. The MH2 domain repeats three times and has homology to the polypyrimidine tract-binding motif of heterogeneous nuclear RNP I/L. NP220 also has a DNA-binding domain and nine repeats of the sequence LVTVDEVIEEEDL (acidic repeat). We have now isolated mouse equivalents of NP220 (mNP220s) and found that NP220s form a family of proteins with four members produced by alternative splicing of a common pre-mRNA. Two longer forms (NP220alpha and NP220beta) have all functional domains mentioned above while two shorter forms (NP220gamma and NP220delta) lack the DNA-binding domain and the acidic repeat. The structural aspects of NP220s are distinct from that of the SR proteins but rather resemble U2AF and Tra2 which activate a specific 3'-splicing site of specific genes in response to differentiation-dependent signals.