Aims: The aim of this study was to evaluate the impact of supplementation by diammonium phosphate (DAP) on hydrogen sulfide (H2S) production, when DAP given either prior to fermentation or during the early stationary growth phase of yeast. Methods and Results: Three contrasting Saccharomyces cerevisiae wine strains were used to ferment synthetic grape juice (GJ) containing 67 mg l-1 of initial yeast assimilable nitrogen (YAN), supplied either as DAP or as mixture of amino acids. Sufficient DAP was added either prior to or 72 h after the initiation of fermentation to achieve a final YAN concentration of 267 mg l-1. Supplementation prior to fermentation stimulated H2S production. The results obtained in model solutions were validated using natural GJ. Conclusion: The timing of DAP supplementation is critical for ensuring that fermentation proceeds without excessive release of H2S. Significance and Impact of the Study: This result has important implications for the wine-making industry, because it highlights the value of determining the initial nitrogen level of a GJ. It raises awareness of the dependence of wine quality on the correct timing of DAP supplementation.