In most neurodegenerative disorders, distinctive intracellular inclusion bodies are found in degenerative neurons, which are known to be neuropathological hallmarks of diseases. Recently, TAR DNA-binding protein of 43 KDa (TDP-43) has been identified as a major constituent protein of ubiquitin-positive inclusions in brains with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). These disorders are now referred to as TDP-43 proteinopathy. TDP-43 deposited in brains with FTLD and ALS was found to be phosphorylated and ubiquitinated. To study the role of these posttranslational modifications in the formation of TDP-43 aggregates, we have produced polyclonal and monoclonal antibodies specific for TDP-43 phosphorylated at Ser409 and Ser 410. These antibodies specifically recognized abnormally phosphorylated TDP-43, but not normal TDP-43 in immunohistochemical analyses of brains of FTLD and ALS patients. Immunoblot analyses using these antibodies showed that phosphorylated and fragmented TDP-43 was deposited in diseased brains. Furthermore, we identified casein kinase 1 as a candidate protein kinase, which was responsible for abnormal phosphorylation of TDP-43. Phosphorylated recombinant TDP-43 proteins were demonstrated to be easier to fibrillate than wild-type TDP-43 in vitro. Recent discoveries of the missense mutations in the TDP-43 gene in familial or sporadic ALS cases prove a direct link between altered TDP-43 function and neurodegeneration. Elucidating the biochemical processes responsible for phosphorylation, fragmentation, and intracellular aggregation of TDP-43 may provide important insights into the pathogenesis of TDP-43 proteinopathy.