The C-terminal propeptide of tobacco (Nicotiana tabacum) chitinase A has been shown to be necessary and sufficient for targeting of chitinases to the plant vacuole. The sequence specificity of this vacuolar targeting peptide (VTP) has now been analysed using transient expression of chitinases in Nicotiana plumbaginifolia protoplasts. An extracellular cucumber chitinase, previously used as a secreted reporter protein in transgenic tobacco, was also secreted into the incubation medium by the transiently transformed protoplasts. Addition of six to seven amino acids at the C-terminus to generate the VTP of tobacco chitinase A were sufficient to cause retention of most of the cucumber chitinase within the protoplasts. The chitinase A itself, as well as a mutant lacking the N-terminal chitin-binding domain, were retained to 80% in the protoplasts when low concentrations of the plasmid were used in the transient expression system. At high concentrations of plasmid, causing high levels of transiently expressed chitinase, retention was reduced, indicating saturation of the sorting system. Deletion of the C-terminal methionine did not affect the intracellular location, but deletion of even a single internal amino acid of the VTP caused predominantly secretion of tobacco chitinase A. In contrast, exchanges of amino acids in the VTP as well as substitution of the VTP with random sequences had intermediary effects that covered the whole range from retention to secretion. The results suggest that the sorting system responsible for the diversion of secretory proteins to the vacuole has a low specificity for the sequence of C-terminal targeting peptides, and that sequence changes in the VTP allow a gradual transition from vacuolar retention to secretion.