Affinity labelling with aldehyde-containing analogs of initiation substrates of nuclear fraction of tick-borne encephalitis virus (TBEV) infected cells results in a labelling of a single polypeptide with a molecular mass of 68 kDa which was immunologically identified as TBEV NS3 protein. A single-hit hydroxylamine hydrolysis, using limited and long-term CNBr cleavages allowed one to identify Lys1800 and/or Lys1803 as the label attachment sites. These amino acid residues are situated in the proximity of the ‘B’-site of NTP-binding motif of viral RNA replicase.