Monoclonal antibody McAb2C9 against Staphylococcal nuclease (SNase R) and its N-terminal fragments was produced and characterized. It was observed that the intact enzyme SNase R and its seven fragments (SNR141, SNR135, SNR121, SNR110, SNR102, SNR79 and SNR52) differed in their interactions with McAb2C9. However, the fragments with weak immunoreactivity, such as SNR141 and SNR110, increased ability reacting with McAb2C9 in their partially unfolded state. It suggests that the differences of immunoreactivity among the fragments are due to diverse extent of the exposure of the specific epitope and the conformation of the peptide fragment. The monoclonal antibody McAb2C9 could be a useful probe to investigate the mechanism of folding of SNase R and its N-terminal fragments.