AimsTo establish a proper protoplast-preparation route from enriched motile flagellates and nonmotile resting cells of Haematococcus pluvialis. Methods and ResultsThrough cultivations in two mixotrophic media, enriched Haematococcus flagellates and resting cells were respectively produced and applied in enzymatic protoplast preparations. Great differences of enzymatic sensitivity and osmotic-lability were identified between them. Flagellates showed the same osmotic-lability as protoplasts and the extracellular matrix-removing rate was applied for an evaluation of protoplast-releasing. During the treatment of flagellates, an addition of more than 02mmoll(-1) Ca2+ was found to be essential for maintenance of high cellular viability. More than 80% cellular viability and a 90% protoplast-releasing rate were obtained simultaneously after 2-3h treatment of 006% proteinase K in 005moll(-1) Tris-HCl (pH78) buffer with 02mmoll(-1) CaCl2 and 02moll(-1) sorbitol/mannitol. For resting cells, a treatment of both 012% proteinase K and a combination of 2% cellulase+1% snailase could function similarly in order to degrade the cellulosic cell wall, while the protoplast yield was limited to about 40%, due to the existence of an undegradable secondary wall in the mature resting cell. ConclusionProteinase K was efficient for protoplast-releasing from either flagellates or resting cells. Due to the great difference of enzymatic sensitivity and osmotic-lability between flagellates and resting cells, it was necessary to select a different enzymatic treating process based upon the main cell type in the culture. A better protoplast-preparing efficiency was obtained from Haematococcus cells when flagellates were in the majority. Significance and Impact of the StudyThe protoplast preparation of H. pluvialis was firstly established based on two main cell types of H. pluvialis, motile flagellates and nonmotile resting cells. Increase of flagellate stability and viable protoplast-preparing efficiency through addition of Ca2+ in enzymatic solution was firstly reported.