Subtilisin Carlsberg from Bacillus licheniformis has been studied by one-dimensional proton nuclear magnetic resonance spectroscopy. The Carr-Purcell-Meiboom-Gil spin echo pulse sequence has been used to record the proton NMR spectra in the aromatic region. Effects of removing bound Ca2+ ions from the enzyme by ethylenediaminetetraacetic acid(EDTA), of binding the inhibitor, phenylmethylsulfonyl fluoride (PMSF), by the active site Ser 221, and of adding KCl or CaCL2 have been investigated. The observed changes in histidine C-2H resonances can be interpreted in terms of the recent high-resolution crystal structure an-alyses.