Transcription antitermination by N proteins of lambdoid phages involves specific interactions of the C-terminal domain of N with the elongation complex (EC). The interacting surface of N on the EC is unknown, knowledge of which is essential to understand the mechanism of antitermination. Specific cleavage patterns were generated near the active site Mg2+of the RNAP of an N-modified stalled EC using Fe-BABE conjugated to the only cysteine residue in the C-terminal domain of N from a lambdoid phage H-19B. Modification of EC by N also induced conformational changes around the same region as revealed from the limited trypsin digestion and in situ Fe-DTT cleavage pattern of the same EC. These data, together with the previously obtained H-19B N specific mutations in RNAP, (G1045D) and ’ (P251S, P254L, G336S and R270C) subunits, suggest that the active center cleft of the EC could be the site of action of this antiterminator. H-19B N-induced altered interactions in this region of EC, prevented the backtracking of the stalled EC at ops pause site and destabilized RNA hairpin-subunit flap domain interactions at the his pause site. We propose that the physical proximity of the C-terminal domain of H-19B N to the active center cleft of the EC is required for the process of transcription antitermination and it involves both stabilization of the weak RNA:DNA hybrid at a terminator and destabilization of the interactions of terminator hairpin in the RNA exit channel.