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Two types of protoplasts were isolated from leaves of shoots or callus of subcultures of jojoba (Simmondsia chinensis (Link) Schneider). Protoplasts from leaves were rich in chloroplasts and were about half the volume of protoplasts from callus. The viability of preparations as determined by the Evans blue technique was 80%. From cell cycle analysis by flow cytometry of nuclei, leaf protoplasts were uniformly in a non-proliferating phase (G0-G1), while callus protoplasts presented many phases of the cell cycle. Protoplasts from calli had only half the Chl of those from leaves. Yet Chl a/b ratio, as well as protein and total lipid content per cell, were similar in both types of protoplasts. A major drop in polar lipids, chiefly in mono- and digalactosyldiacylglycerol, and a parallel increase in neutral lipids occurred during protoplast isolation. The 18:2/18:3 ratio decreased in neutral lipids concomitant with an increase in triglycerides rich in linolenic acid. Our results suggest a triggering of lipolytic acylhydrolases during the protoplast isolation, as reported for other species. Plasmolysis of the cells with high osmolarity medium and long incubation times were required to get a good yield of jojoba protoplasts. In the course of this procedure water-deficit stress takes place. A parallel with lipid changes occurring under this type of stress is discussed.