The hygromycin B phosphotransferase gene (hpt) has been widely used in the process of plant genetic engineering to produce plants that can secrete the HPT protein. As part of a safety assessment, sufficient quantities of the protein were produced in Escherichia coli to conduct in vitro digestibility and animal studies. Western blotting analysis showed that the HPT protein was digested by simulated gastric fluid within 40 s. ELISA demonstrated that the protein did not induce detectable levels of specific IgE antibodies or histamine in test animals. Alignment of the amino acid sequence of HPT with those of known allergens did not produce evidence of sequence similarities between these allergens and the HPT protein. We conclude that HPT has a low probability to induce allergenicity.