The single histidine residue (His-15) in hen egg white lysozyme (EC 3.2.1.17) was chemically modified by diethyl pyrocarbonate (DEPC) to form exclusively the mono-N-carbethoxyimidazole adduct (second order rate constant of 252 +/- 16 M-1 min-1). Irreversible biscarbethoxylation of the His-15 imidazole ring by DEPC was observed when lysozyme was pretreated with 2-mercaptoethanol (2-ME), 2-ME plus 8 M urea, or 2-ME plus 1% (w/v) sodium dodecyl sulfate (SDS). Circular dichroism difference spectra were measured for the mono-N-carbethoxyimidazole derivatives of lysozyme, N alpha-acetyl-L-histidine, angiotensin-II, and O-carbethoxy-N alpha-acetyl-L-tyrosine. The circular dichroism difference spectrum for mono-N-carbethoxy lysozyme had one main band (delta [theta]244 nm = +17,000 degree. cm2.dmol-1) in the 240-260 nm region. Denaturing mono-N-carbethoxy lysozyme with 2-ME and 8 M urea (55 degrees C) or 1% SDS (100 degrees C) essentially abolished its circular dichroism difference spectrum in the 240-260 nm region without any decarbethoxylation. In this same region the circular dichroism difference spectra of DEPC-modified N alpha-acetyl-L-histidine and DEPC-modified angiotensin-II had two much weaker bands (delta [theta]233 nm = +1000 degree.cm2.dmol-1 and delta[theta]252nm = -600 degree.cm2.dmol-1 for N alpha-acetyl-L-histidine). This study reports the first characterization of circular dichroism associated with mono-N-carbethoxyhistidine in an enzyme (lysozyme), a peptide (angiotensin-II), and a model compound (N alpha-acetyl-L-histidine).