Though fast atom bombardment ionization makes possible the ionization and molecular weight determination of polar or thermally labile biological compounds, the resulting mass spectra commonly give few or no fragment ions which would allow detailed structural analysis. In particular, isomeric compounds often give identical spectra. Collision-induced dissociation of ions resulting from fast atom bombardment ionization is shown to be a powerful combination which can differentiate isomeric substances. The technique is applied to isomeric bile acid salts and steroid conjugates and is capable of differentiating structural isomers which have similar fast atom bombardment mass spectra. A range of isomeric cyclic nucleotides is also shown to be amenable to the method. Sensitivity limits are examined and the unequivocal identification of two 3′,5′-cyclic nucleotides isolated from living systems is demonstrated.