The pentapeptide with the sequence Gln-Tyr-Asn-Ala-Asp (QYNAD) was isolated from cerebrospinal fluid (CSF) of patients with inflammatory demyelinating neurological diseases. QYNAD was shown to inhibit Na⁺ currents by shifting the steady-state inactivation curve to more negative potentials. However, this finding was not consistently reproducible in other laboratories. This led to the following hypotheses. First, the efficacy of QYNAD could depend on certain conditions of peptide synthesis or on the presence of still unknown cofactors in CSF. Second, QYNAD could be one factor among a group of related peptides that are targets of enzymatic degradation and modification. The possible influence of cofactors and by-products on the biological activity of QYNAD was studied by whole-cell recordings on HEK 293 cells functionally expressing rat Nav1.2 channels. Further, the possible loss of an additional amino acid that may have occurred during storage of CSF or peptide isolation was investigated. Using highly purified peptides, we did not observe significant effects of QYNAD (100 μM) on Na⁺ currents. Permutated peptides (YNQDA), peptides of altered length and charge, tetra- and tripeptides derived from the sequence had no or only slight effects on Na⁺ currents. Only peptide mixtures with the sequence XQYNAD (where X is one of 20 L-amino acids) caused a reversible block of rat Nav1.2 channels due to a shift of voltage dependence of activation. We used this peptide mixture, XQYNAD, as a model to study the possible enzymatic degradation of peptides and their modification in CSF. Peptide concentrations were determined by mass spectrometry using an LC/MSD Trap system 1100 Series (Agilent, Germany). Incubation of XQYNAD (100 μM) with 300 μl samples of CSF at 37°C resulted in a release of QYNAD (MW: 609 Da) within several hours and a further conversion to its pyroglutamic acid form (pyQYNAD, MW 592 Da). After 5 h incubation the average QYNAD concentration was 6.6 ± 2.2 μM (n = 27), while the pyQYNAD concentration yielded 5.19 ± 1.86 μM. In the presence of the low MW (< 3 kDa) fraction of CSF neither QYNAD was released nor was the conversion of synthetic QYNAD to pyQYNAD observed. To study the conversion of QYNAD to its pyroglutamic acid derivative more exactly, we incubated QYNAD (10 μM) with CSF samples. Under control conditions 83?90% were converted to pyQYNAD within 5 h. This conversion could be prevented by inhibitors of the enzyme glutaminyl cyclase (EC 2.3.2.59). The addition of benzylimidazole (35.5 or 71 μM) or acetlyhistamine (85.5 or 170 μM) to the samples led to a decrease of pyQYNAD to 38-60% (n = 8) of total detected peptide. We conclude that small peptides underlie dynamic processing in CSF and that these processes are dependent on the high MW fraction of CSF, probably on the activity of an amiopeptidase. The fact QYNAD is converted to its pyroglutamic acid derivative in CSF indicates the presence and activity of glutaminyl cyclase. To clarify the role of QYNAD and related peptides in the pathogenesis of inflammatory demyelinating neurological diseases it will be helpful to study enzymes that degrade and modify peptides in the CSF.