The 2H and 13C enrichment, 13C–13C coupling patterns and β-2H isotope shifts observed in β-barbatanol prepared from (–)-β-barbatene incorporating variously 2H- and 13C-labelled mevalonates, acetates and glycerol verifies a 1,4-hydrogen shift and a double 1,2-methyl migration in the formation of β-barbatene in cultured cells of Heteroscyphus planus, and also indicates the diversity of regulation and the sole operation of the mevalonate pathway in extrachloroplastidic sesquiterpene biosynthesis.