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Carotenoids are involved in a variety of biological functions, yet the underlying mechanisms are poorly understood, in part because of the long-standing difficulty in assigning the location of the first excited (S₁) state. Here, we present a method for determining the energy of the forbidden S₁ state, on the basis of ultrafast spectroscopy of the short lived level. Femtosecond transient absorption spectra and kinetics of the S₁ → S₂ transition revealed the location of the intermediate level in two carotenoid species involved in the xanthophyll cycle, zeaxanthin and violaxanthin, and yielded surprising implications regarding the mechanism of photoregulation in photosynthesis.