The objective of present work was to asses the possibility of the protoplast formation of Rhodotorula glutinis completely by enzymatic method. The main lytic enzyme to hydrolyzed the Rh. glutinis cell was identified as mannanase and protease in the induced exoenzyme of F-5 fungus isolated from soil. Penicillium wortmanni having high mannanase activity was selected, and induced exoenzyme of this fungus was able to promote the lysis of the fractionated cell wall and Rh. glutinis intact cell in growing phase. Among the carbon substrates selected to induce mannanase from P. wortmanni, AWY gave more excellent production than any other carbon sources examined, even though cellulose, glucose, and mannose can be used as carbon source for lytic enzyme production. In lytic system of P. wortmanni, mannanase, $\beta$-galactosidase, and $\beta$-mannosidase were separated by Sephadex G-100-50 column chromatography. Cell lysis was observed up to 80\% in the TED-pretreatment cell suspension with reconstituted lytic enzyme system. This effect may be due to the hydrolysis of the fucogalactomannan sieve, the outer layer of cell wall, to be loosened by the pretreatment of dithiothreitol, and then, the degradation of glucomannan structure responsible for the rigidity of the cell wall. Mannanase from P. wortmanni had the optimum condition at $30\,^\circ\!C$, acidic pH.