We have modified the 3'-coding region of the hygromycin B (Hy) phosphotransferase-encoding gene (hph). The level of Hy resistance conferred by the modified hph genes was determined in Escherichia coli and Nicotiana tabacum. Polypeptides up to 12 amino acids shorter or longer than the wild-type (wt) protein remained active in both pro- and eukaryotic cells. A gene construct of wt length, but with a modified 3' sequence, was less active as measured by the level of Hy resistance. Therefore, the nature of the C terminus of the hph gene product, rather than the length of the polypeptide, influences the ability to confer resistance to Hy.