We have previously reported that a recombinant strain of Serratia marcescens was constructed from a host released from feedback repression of biotin biosynthesis and a recombinant plasmid carrying the mutated biotin operon. To improve D-biotin production, we constructed a recombinant plasmid pLGM304PA having the mutated biotin operon, a plasmid-stabilizing element and the ampicillin resistant gene, and introduced it into the D-biotin producing strain ETA23K-8 which contained an additional copy of the mutated biotin operon and the kanamycin resistant gene, resulting in ETA23K-8(pLGM304PA). Studies of the growth conditions for this recombinant strain indicated that high concentrations of sulphur and ferrous iron were required for good production of D-biotin. A batch culture fed by continuous addition of sucrose gave a maximum production of 600 mg litre(-1).