Five geometrical isomers of lycopene were isolated from a photoisomerized mixture on a semi-preparative C30 column and characterized by UV-vis spectroscopy, mass spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. 1H NMR and 2D NMR measurements were used to unambiguously assign the double bond configuration of five isomers: (5Z,9'Z)-, (9Z)-, (5Z,9Z)-, (all-E)-, and (5Z)-lycopene. This will allow other laboratories to use the results for their C30 HPLC. In addition, it is possible to investigate structure-activity relationships for these lycopene isomers which will improve the understanding of their physiological role in biological tissues.