Clavulanic acid is a naturally occurring antibiotic produced by Streptomyces clavuligerus. The present work reports on clavulanic acid production by Streptomyces clavuligerus MTCC 1142 using one-factor-at-a-time and L(25)orthogonal array. The one-factor-at-a-time method was adopted to investigate the effect of media components (i.e., carbon source, nitrogen source and inoculum concentration) and environmental factors such as pH for clavulanic acid production. Production of clavulanic acid by Streptomyces clavuligerus was investigated using seven different carbon sources (viz. glucose, sucrose, modified starch, rice-bran oil, soybean oil, palm oil, and glycerol) and six different nitrogen sources (viz. peptone, yeast extract, ammonium chloride, ammonium carbonate, sodium nitrate and potassium nitrate). A maximum yield of 140 mu g/ml, clavulanic acid was obtained in the medium containing soybean oil as a carbon source and yeast extract as nitrogen source. Subsequently, the concentration of soybean flour, soybean oil, dextrin, yeast extract and K2HPO4 were optimized using L-25 orthogonal array method. The final optimized medium produced 500 mu g/ml, clavulanic acid at the end of 96 h as compared to 140 mu g/mL before optimization. Synthesis of precursor molecules as a metabolic driving force is of considerable importance in antibiotic synthesis. Attempts to increase the clavulanic acid synthesis by manipulating the anaplerotic flux on C-3 and C-5 precursors by supplementing the medium with arginine, ornithine, proline, valine, leucine, isoleucine, pyruvic acid and alpha-ketoglutarate were successful. Supplementing the optimized medium with 0.1 M arginine and 0.1 M leucine increased the yield of clavulanic acid further to 1100 mu g/mL and 1384 mu g/mL respectively.