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Mutations in the antioxidant enzyme superoxide dismutase-1 (SOD1) are a well-established cause of amyotrophic lateral sclerosis (ALS). The mutations promote SOD1 misfolding, resulting in protein aggregation and motor neuron degeneration. SOD1 is normally a structurally stable enzyme, and the mechanisms underlying SOD1 misfolding remain poorly understood. Approximately one third of SOD1 in cerebrospinal fluid (CSF) exhibits an N-terminal truncation, the biological significance of which remains unclear. This is remarkable given the dramatic effects ALS-linked C-terminal truncations have on the enzyme. In this study, we identified the truncation site and investigated its impact on SOD1 stability and enzymatic activity. Edman degradation revealed the cleavage site between Asn-26 and Gly-27, generating a 26-residue peptide that was confirmed by mass spectrometry. We analyzed postmortem tissues from different parts of the central nervous system (CNS), including the choroid plexus, and found only trace amounts of N-terminally truncated SOD1. Biochemical characterization of the SOD1 in CSF was done by size exclusion chromatography, ion exchange chromatography, and mass spectrometry. Our findings demonstrate that SOD1 in CSF retains full enzymatic activity, that the N-terminally truncated variant is mainly present in heterodimers with native SOD1 subunits, and that the dimer remains folded and active, with both fragments of the truncated SOD1 fixed after proteolysis. Truncated SOD1 was absent in human plasma. In mice, only transgenically expressed human SOD1 underwent truncation in CSF, whereas endogenous murine SOD1 remained intact. Lastly, the N-terminal truncation does not induce misfolding, unlike the destabilizing effects observed with C-terminal truncations. The location where the truncation takes place and the underlying mechanism could not be identified. Whether the N-truncated SOD1 variant contributes to ALS pathogenesis remains to be determined. (Figure presented.).