The reversible cis/trans photoisomerization of secondary thiopeptide bonds has been systematically studied with UV-visible absorption, capillary electrophoresis, 1H NMR spectroscopy, and circular dichroism methods. It was found that the concentration of the cis conformers could be increased from less than 1 % in the thermal equilibrated solution to up to 20 % in the photostationary state. The rotational barriers of the thiopeptide bond and the pH dependence of the isomerization rates were also studied. The quantum yields of the trans-->cis and the cis-->trans processes were determined from photokinetic analysis.