The addition of glycerol, ornithine, and arginine had significant effects on the biosynthesis of clavulanic acid in Streptomyces clavuligerus. Adding ornithine in batch culture could enhance clavulanic acid production but arginine showed little effect. With an addition of 5 mM ornithine to the culture, the CA production (200 mg l(-1)) was about 2-fold of that without any addition of amino acids (115 mg l(-1)). In the fed-batch experiments, feeding glycerol could raise clavulanic acid production to 220 mg l(-1). In the absence of glycerol, feeding ornithine or arginine only could not enhance the clavulanic acid production. We suggested that glycerol (C-3) supply rather than amino acid (C-5) supply is rate-limiting. Feeding glycerol with ornithine resulted in further increase in clavulanic acid production to 311 mg l(-1), but feeding glycerol with arginine showed little enhancing effect. Feeding ornithine not only provided an enough supply of arginine for clavulanic acid production, but also inhibited the glycerol-utilizing cephamycin biosynthesis. Thus, it could increase the flux of C-3 precursor into clavulanic acid. The results reveal that ornithine rather than arginine could enhance effectively clavulanic acid production if the amount of C-3 precursor was sufficient.