The yield, viability and continuous culture of isolated Italian ryegrass protoplasts were investigated. The effects of cold treatment (4^{circ}C.$) for 7 days and basic LS medium supplemented with 5mg/l $AgNO_3$ showed effectively on embryogenic callus induction and regeneration responses of immature and mature embryos or young inflorescences subcultured every 4 weeks on basic medium. The optimum combinations of growth regulator on the regeneration responses was 0.2mg/l BAP and 2mg/l 2, 4-D. Calli induced inflorescences were suspended in its liquid medium for 5 days before enzyme treatment. Maximum protoplast yield and viability were obtained after digestion in enzyme solution contained 4% cellulase R10. 2% macerozyme and 2% pectinase in 0.6M mannitol. Cell division and microcalli development were observed in isolated protoplasts cultured in agarose culture of KM8P medium.