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This study describes a protocol for regeneration of plants from cell suspension-derived protoplasts of American elm (Ulmus americana). Efficient protoplast isolation was achieved from a two-phase culture system through the incorporation of 100 μM 2-aminoindan-2-phosphonic acid, with a yield of approximately 2 × 10(6) protoplasts/ml packed cell volume. Isolated protoplasts failed to survive in liquid or alginate bead culture systems but initiated and continued to divide when embedded in low melting point agarose beads. Protoplast-derived callus proliferated and differentiated into shoot buds in response to 10 or 20 μM thidiazuron. Differentiated buds elongated and continued to proliferate on elm shoot medium supplemented with 3.0 μM GA3. The protoplast-derived shoots rooted and acclimatized to greenhouse conditions and continued to grow. This system provides the first protoplast-to-plant regeneration system for American elm and provides a framework for the development of protoplast fusion or genome editing technologies.