Neurofilament (NF) aggregates in motor neurons are a key neuropathological feature of amyotrophic lateral sclerosis (ALS). We have previously observed an alteration in the stoichiometry of NF subunit steady state mRNA levels in ALS spinal motor neurons using in situ hybridization and proposed that this led to aggregate formation. We have now examined the levels of NF mRNA in whole tissue homogenates of spinal cord using the RNase protection assay and real time reverse transcriptase-PCR and observed significant elevations of NF mRNA level in ALS. Compared with age-matched control, we observed a greater stability of heterogeneously expressed NFL mRNA in the presence of ALS spinal cord homogenates. Heat denaturing or protease K digestion of the control homogenates increased the stability of the NFL mRNA to levels observed in ALS homogenate. Increased NFL mRNA stability was also induced by increasing the percentage of ALS homogenate in an admixture of control and ALS homogenates. These observations suggest the presence of trans-acting NFL mRNA-destabilizing elements in control but not in ALS spinal cord homogenates. This was confirmed in gel retardation assays. We also observed that the destabilizing elements interact with the 3'-untranslated region of NFL mRNA. These findings suggest that the trans-acting NFL-destabilizing elements are selectively suppressed in ALS homogenates, resulting in an increased stability and level of NFL mRNA.