The interaction of biological carotenoids with 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD), a thermodissociable source of electronically excited ketones, was investigated using reversed-phase high-performance liquid chromatography. Incubation of the all-trans isomers of beta-carotene, lycopene and canthaxanthin with HTMD led to significant trans-to-cis isomerization, with cis isomers accounting for 20-50% of products formed (the balance assigned as oxidation products). The isomers forming from all-trans-beta-carotene were identified as 9-cis-, 13-cis- and 15-cis-beta-carotene by cochromatography of cis isomer standards and by on-line diode array absorbance spectroscopy. An HTMD-dependent cis-to-trans isomerization was observed in incubations started with 15-cis-beta-carotene, and it occurred more rapidly and to a greater extent than the isomerization of all-trans-beta-carotene. The isomer patterns generated from lycopene and beta-carotene are generally similar to those reported recently for various human tissues (Stahl et al., 1992, Arch. Biochem. Biophys. 294, 173-177).