Polyribosomes (polysomes), active in an amino acid incorporation system in vitro, were isolated from tobacco leaf protoplasts. A comparison of polysome profiles indicated that the polysome/monosome ratio is greatly decreased in isolated protoplasts as compared to the intact leaf. In isolated protoplasts, a marked accumulation of ribosomal subunits was also found. The division of protoplasts, as investigated in the 8-cell and callus stages, was associated with a(n) (at least) partial regeneration of polysome profiles characteristic for leaves. Plasmolysis of leaves attached to the plant had no great effect on the polysome profile. However, leaf excision per se resulted in a dramatic loss of polysomes, even when the leaf tissue was floated on water. It is concluded that the isolation of the cell from its normal environment, and not the osmotic stress and associated increase in RNase activity, is the most important factor responsible for the loss of polysomes in isolated protoplasts.