Methods were developed for the isolation of large numbers of healthy protoplasts from two species of the agarophyte Gracilaria; G. tikvahiae McLachlan and G. lemaneiformis (Bory) Weber‐van Bosse. This is the first report of protoplast isolation and cell division in a commercially important, phycocolloid‐producing red seaweed, as well as for a member of the Florideophycidae. The optimal enzyme composition for cell wall digestion and protoplast viability consisted of 3% Onozuka R‐10, 3% Macerozyme R‐10, 1% agarase and 0.5% Pectolyase Y‐ 23 dissolved in a 60% seawater osmoticum containing 1.0 M mannitol. The complete removal of the cell wall was confirmed by several different methods, including electron microscopic examination, and the absence of Calcofluor White (for cellulose) and TBO (for sulfated polysaccharide) staining. Spontaneous protoplast fusion was observed on several occasions. Protoplast viability was dependent upon the strain and age of the parent material, as well as the mannitol concentration of the enzyme osmoticum. Cell wall regeneration generally occurred in 2‐6 days; cell division in 5‐10 days. Protoplast‐produced cell masses up to the 16‐32 cell stage have been grown in culture. However, efforts to regenerate whole plants have been unsuccessful to date.

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