Haploidization in onion (Allium cepa L.) using immature flower buds simulates zygotic embryogenesis with no fecundation. In order to know the involvement of polyamines (PAs) in this process, we determined the concentration of endogenous PAs in flower buds and experimented the addition of various combinations of PA molecules in the medium. At the inoculation stage, high levels of free and conjugated spermidine and low putrescine + hydroxyputrescine/spermidine + spermine ratio characterized the highest responsive varieties. During in vitro culture, high levels of putrescine and its derivatives characterized the lowest responsive varieties, whereas high levels of spermidine and spermine characterized responsive varieties. The putrescine + hydroxyputrescine + homospermidine/spermidine + spermine ratio remained low in responsive varieties. The addition of spermidine or spermine (2 x 10-3 M) to the culture medium improved significantly the embryo production. Our results suggest that the arginine decarboxylase pathway is involved in PA biosynthesis during the in vitro culture of flower buds. Our study showed that specific ratios of PAs are required for successful gynogenesis in onion.