A cell-free extract from Escherichia coli containing an E. coli biotin synthase that was expressed to approx. 1 % of soluble cell protein by cloning the E. coli bioB gene was used to investigate the biotin synthase reaction. The pH optimum was between 8 and 8n5, and the reaction velocity was dependent on the concentrations of dethiobiotin, cysteine, S-adenosylmethionine and asparagine. The catalytic-centre activity of the enzyme in itro was estimated to be 0n95 h −" , and each molecule of enzyme turned over less than one molecule of dethiobiotin, i.e. the enzyme was not acting catalytically. HPLC analysis of reaction mixtures revealed the presence of a compound with the characteristics of an intermediate : (1) it was labelled with "%C, and therefore derived from the ["%C]dethiobiotin substrate ; (2) it was present only in reaction mixtures containing biotin synthase ; (3) it was not derived from ["%C]biotin ; (4) $&S from [$&S]cystine was