Polyamines, putrescine, spermidine and spermine are low molecular weight polycations implicated in flowering and seed set and plants’ responses under adverse environmental conditions. Research in other crops has shown that polyamine metabolism is greatly affected by water-deficit stress, however, no information exists on cotton (Gossypium hirsutum L.). A field study was conducted in 2011 in two contrasting locations (Fayetteville, AR, and Lubbock, TX) in order to investigate the effect of water-deficit stress during flowering on polyamine metabolism of the cotton flower and its subtending leaf. Treatments consisted of control (well watered) and water-stress (irrigation withheld for two weeks at the onset of flowering) in a split plot design. First day white flowers and their subtending leaves were collected at the end of each week of the stress period for determination of polyamine concentrations. Water-deficit stress resulted in significant increases in PUT and SPD levels of pistils and leaves compared to the control. However, pistil and leaf SPM content significantly increased under drought conditions in one location and remained unaltered in the other one. Leaf and pistil polyamine metabolism of cotton appeared to be greatly affected by limited water supply, however further research is needed to elucidate the ways polyamines can be used to increase cotton drought tolerance.