The amount and timing of nitrogen (N) application to a vineyard is critical for must yeast assimilable nitrogen (YAN) concentrations. YAN concentrations and amino acid profiles are important for the fermentation process and wine composition. Commonly, N is applied at flowering to optimize leaf functioning or after harvest to enhance vine productivity the following season. In this study N was applied at various stages of berry development to determine allocation patterns between vine perennial and annual components and to assess when berry YAN concentrations can best be optimized. Five year old potted 'Chardonnay' vines received ammonium sulfate fertilizer at six different times from full bloom to two weeks before harvest and were also exposed to either full or half irrigation during that period. Reduced water supply resulted in a higher allocation of N to the perennial structures and less to the annual components of the vine. N allocation to the annual components of the vine was greatest when it was applied at full bloom, however allocation to the perennial components was greatest when it was applied after fruit-set to veraison. The timing of N supply had a substantial influence on YAN concentrations, and was highest when N was applied about two weeks after veraison. Low water supply also resulted in higher juice YAN concentrations. The perennial N reserves in the roots were highest under low water supply and when N was applied at veraison, while the allocation to the annual parts was lower under this irrigation regime. The study indicates that timing of N application and the application of water constraints during berry development can impact on N partitioning, while the total amount accumulated by the vine is not altered.