The perfusion culture technique using a shaken ceramic membrane flask (SCM flask) was applied to achieve high-level expression of recombinant gene. A recombinant methylotrophic yeast strain, Pichia pastoris, was cultured aerobically with head space ventilation on a reciprocal shaker using an SCM flask. High-level production of beta-galactosidase was attempted by increasing both the cell concentration and the intracellular content of beta-galactosidase. The productivity and yield of beta-galactosidase were compared between two-stage culture and growth-associated production methods, In the two-stage culture method, the cell concentration was first raised to 57 g/l at 121h by feeding glycerol and, thereafter, expression of beta-galactosidase was induced by feeding methanol. The beta-galactosidase activity in the culture broth quickly increased up to 96 kU/ml within 48 h of initiating the production phase, while the cell concentration continued to increase, reaching 106 g/l after 167 h culture. On the other hand, in the growth-associated production method, beta-galactosidase was produced from an early stage of the culture by the feeding of methanol. The beta-galactosidase activity reached 152 kU/ml at 168 h, while the cell concentration was depressed to 49.7 g/l. The results showed that the growth-associated production method with the feeding of methanol was highly effective for high-level expression of the methanol-inducible recombinant gene of Pichia pastoris. A beta-galactosidase productivity level 10 times higher than that obtained in an ordinary fed-batch culture using a shake flask was readily achieved by continuous replenishment of the culture supernatant.