The concentrations of free and non-free spermidine and spermine in vegetative (leaves, shoot apices) and reproductive (lateral buds, ovaries, fruit tissues) olive organs were determined. The aim was to investigate whether the seasonal fluctuations of these polyamines in different organs correlated to developmental processes such as floral differentiation, shoot growth, anthesis, fertilisation, and fruit growth. Samples were taken from trees of the large-fruited cultivar Chondrolia Chalkidikis and the small-fruited Koroneiki. High levels of both polyamines were determined in buds and shoot apices during the cold period, especially in Ch. Chalkidikis, possibly connected to cold-hardiness exhibited by this cultivar. Two peaks of total spermine in buds, the first occurring in January–February and the second in mid April, coincided with the morphological differentiation of flowers and the inflorescence emergence, respectively. Polyamine levels in ovaries dropped after ovule fertilisation and remained low during the subsequent growth of fruit tissues. Spermidine accumulation, especially in the non-free form, was recorded around the middle of August during a period of intense physiological activity in olive connected to embryo and fruit growth, endocarp hardening, and preceding the second flush of growth in early September. The data showed a direct relationship, in terms of time, of polyamine fluctuations with developmental processes in olive. Whether polyamines act, in this respect, as nitrogen providers or assume a more active regulatory role remains to be elucidated.