Recently we are interested in the biosurfactant. Biosurfactant have a low toxcity and easily biodegradable compound. Pseudomonas sp. 13 was isolated from soil. This microorganism produced biosurfactant that consists of glycolipid R-1 and R-2. A time course study of fermentation indicated that the appearance of glycolipid in the fermentation broth the commencement of the stationary phase with the respect to biomass. The effect of variation of the media components such as amount of glucose, nitrogen, phosphate and metal ions has been investigated The following values found to be optimum for biosurfactant production (glucose, 20 g/ℓ; carbon to nitrogen ratio, 40; carbon to phosphate, 18; FeSO₄·7H₂O 20 ㎎/ℓ).