Three 2,3,22,23,24‐pentahydro‐21H‐biline‐1,19‐dion‐10‐enium salts have been prepared: 17,18,22,23‐tetramethyl‐2,3,22,23,24‐pentahydro‐21H‐biline‐1,19‐dion‐10‐enium methanesulphonate (1) with a central flexible structure, its 10‐syn‐locked 22,23‐methylene homologue (2) with a central Z‐syn structure and its 8,12‐isopropylidene homologue (3), the first bile pigment with a central E‐anti structure. For comparison, the corresponding E‐anti 22,23,24‐trihydrobilinedionium salt 3a was also prepared. The 2,3 single bond induces a more delocalized charged system, having higher fluorescence quantum yields than observed for the fully conjugated systems. The flexible systems 1, 1a, which adopt an E‐anti conformation, do not show fluorescence. The E‐anti systems have a larger Stokes shift than do the Z‐syn systems. In the pentahydrobilinedionium salts, the highest positive charge resides on the central C10 atom as apparent from 1H NMR spectroscopy and as was corroborated by the finding that the site of nucleophilic attack is invariably at C10. The pentahydro salts also undergo electrophilic H‐D exchange at positions 5, 7 and 12. From the spectral data presented it would appear that the chromophore in (allo)phycocyanine will occur as a protonated system in a rigid protein environment in the E‐anti conformation.