The scaffold attachment factor A (SAF-A) is an abundant component of the nuclear scaffold and of chromatin, and also occurs in heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. Evidence from previous experiments had suggested that SAF-A most likely has at least two different functions, being involved both in nuclear architecture and RNA metabolism. We now show that the protein has a novel scaffold-associated region (SAR)-specific bipartite DNA-binding domain which is independent from the previously identified RNA-binding domain, the RGG box. During apoptosis, but not during necrosis, SAF-A is cleaved in a caspase-dependent way. Cleavage occurs within the bipartite DNA-binding domain, resulting in a loss of DNA-binding activity and a concomitant detachment of SAF-A from nuclear structural sites. On the other hand, cleavage does not compromise the association of SAF-A with hnRNP complexes, indicating that the function of SAF-A in RNA metabolism is not affected in apoptosis. Our results suggest that detachment of SAF-A from SARs, caused by apoptotic proteolysis of its DNA-binding domain, is linked to the formation of oligonucleosomal-sized DNA fragments and could therefore contribute to nuclear breakdown in apoptotic cells.