Arabidopsis hairy roots were used to produce human gastric lipase. When treated with 2,4-D, the hairy roots developed into thick organs that produced more protein than untreated roots. This was first assessed using green fluorescent protein-producing root lines from which the protein diffused into the culture medium. When growing hairy roots which express the human gastric lipase gene, very little lipase was found in the medium. Incubating the roots in a low pH buffer resulted in lipase diffusion into the buffer, avoiding the need for grinding. The activity of the enzyme on 4-methylumbellireryl-oleate and on tributyrin was determined. Approximately 6000 units of enzyme were recovered per gram of root. The enzyme was also extracted from freeze-dried roots before and after a 2-month storage period at room temperature. This work demonstrates the relevance of Arabidopsis hairy roots for the production of human gastric lipase.