An efficient sporulation/immobilization procedure for immobilized fungal cell culture was developed by modifying an existing immobilized technique to shorten the time and number of steps for sporulation. This method was applied to an immobilized-cell perfusion bioprocess (IPB) for continuous production of CyA, an intracellular secondary metabolite produced by a filamentous fungus, Tolypocladium inflatum. In the IPB, the fungal cells were immobilized in the pores of celite beads (100 similar to 500 mu m) and a top-driven stirred tank fermenter was used for the culture. The IPB showed good process benefits as demonstrated by the high density of immobilized cells continuously producing CyA-containing free cells. The productivity of CyA-containing free cells in the effluent was very high, ca. 1.0 g/(L/h) at a dilution rate of 0.1. h(-1), due to the high density of immobilized cells in the fermenter. The CyA productivity was 4.0 similar to 6.0 mg/(L/h) which was about 6 similar to 10-fold higher than that of batch suspended cell culture. Such an efficient IPB was possible since a decanter was developed in this study, which could effectively separate cell-immobilized beads from the effluent although bead loss slightly increased as the cell loading increased in the latter part of culture. Furthermore, long-term operation of IPB was carried out successfully by employing an in-situ immobilization strategy. It was found that a large number of spores in the fermentation broth in the reactor were entrapped in-situ into the newly supplemented celite beads and then germinated, thus forming new immobilized cells.