Fast atom bombardment mass spectrometry following hydrogen--deuterium exchange in [hydroxy-2H3]glycerol and 2H20 has been studied as a means of establishing active hydrogen content in molecules of unknown structure. Nucleotides, carbohydrates, one peptide and complex antibiotics, in the mass region to 1500 daltons and 29 exchangeable hydrogens were examined, with a correct hydrogen count unambiguously measured in every case. The method is experimentally simple and applicable on a microgram scale, to salts and a variety of polar compounds of biological origin.