We found that the sequences YPLDL and YPLDLF in the large subunit of spinach D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) met the structure YP-aliphatic amino acid which might have opioid activity. We then synthesized these peptides to test their opioid activity. The IC50 of these peptides in mouse vas deferens assay were 51.0 μM and 24.4 μM, respectively, and those in δ receptor binding assay using [3H]deltorphin II as radioligand were 2.09 μM and 0.93 μM, respectively. Both peptides were selective for δ receptor. We named them rubiscolin-5 and -6, respectively. Rubiscolin-5 and -6 have antinociceptive activity in mice after i.c.v. or oral administration. The enzymatic conditions to release rubiscolin were investigated using both spinach Rubisco and synthetic fragment peptides. This is the first example of bioactive peptides derived from plant Rubisco.