Protoplasts were isolated from the primary leaves of lettuce (Lactuca sativa L.) seedlings 10 days after in vitro germination. The leaves were stripped and incubated in an enzyme mixture consisting of 1.2% Cellulase R-10 and 0.3% Macerozyme R-10 in cell and protoplast washing solution (CPW) overnight. The average protoplast yield was 8.25 x 106 protoplasts per g of fresh leaf tissue. When protoplasts were cultured at a density of 3.0 × 105 protoplasts/mL in agarose solid KM8P/KM8 medium, first and second divisions were observed in the protoplasts within a week. Protoplast-derived microcolonies formed after 4 weeks of culture, and visible colonies were present after 3 months of culture. Protoplast-derived microcalli were transferred to Murashige and Skoog medium supplemented with 2.0 mg/L kinetin and 0.1 mg/L NAA and incubated in the light for 3 weeks. They grew into callus, which then regenerated into plants after 7 weeks of culture. The regenerated plants grew as apparently normal flowering fertile plants.