Beauverolides Ja and Ka from Beauveria bassiana strain UICP 22 are cyclotetradepsipeptides: Ja is cyclo-[3-hydroxy-4-methyloctanoyl-L-tryptophyl-L-phenylalanyl-D-alloisoleucyl], and Ka is its 3-hydroxy-4-methyldecanoyl homologue. Their amino acid sequence was deduced from mass spectral analysis, with L-tryptophan confirmed by hydrolysis with toluene-p-sulphonic acid. Pairs of beauverolides differing only in the hydroxy-acid residue were readily separated by high-pressure liquid chromatography (HPLC). Using this technique, beauverolides Ba and Ca were isolated from the binary mixtures Ea+Ba and Fa+Ca for the first time, avoiding the loss of Ba and Ca associated with the previous recrystallisation method.