ABSTRACT Oligopeptides from grape must represent a secondary source of nitrogen for yeasts to grow and carry out fermentation. Saccharomyces cerevisiae takes up oligopeptides from the environment through multiple oligopeptide transporters with different peptide length specificities. However, due to difficulties associated with the qualitative and quantitative measurement of peptides in natural matrices, peptide transporter specificities have been mostly researched in single peptide environments. Using a peptide mapping method, we monitored the relative consumption of peptides derived from a protein hydrolysate by a set of CRISPR-Cas9-engineered S. cerevisiae wine strains to study oligopeptide transporters from the Opt and Fot families. Results show that Opt2 can import peptides containing three to at least seven amino acid residues, which is a broader peptide length specificity than previously reported, while Opt1 activity was not sufficient to support growth on peptides as nitrogen source. Fot1, Fot2, and Fot3, previously referred to as di-tripeptide transporters in S. cerevisiae wine strains, could also import tetrapeptides. The consumption order of peptides was determined by the peptide length as higher chain length peptides were taken up by Opt2 only after most di-tetrapeptides were depleted from the media. Altogether, Fot and Opt2 activity assured completion of the fermentation process without necessarily requiring ammonia or free amino acids. Analysis of peptide transporter gene expression during fermentation showed an effect of SO 4 2− not only on OPT1 but also on FOT and supported the assumption of a possible interplay between Fot and Opt2 activities. IMPORTANCE Limited nitrogen supply can prevent the completion of alcoholic fermentation. Supplementation through peptides as an alternative, natural source of nitrogen for yeast offers an interesting solution for this issue. In this work, the S. cerevisiae peptide transporters of the Opt and Fot families were studied. We demonstrated that Fot and Opt2 have a broader peptide length preference than previously reported, enabling yeasts to acquire sufficient nitrogen from peptides without requiring additional ammonia or amino acids to complete fermentation. On the contrary, Opt1 was unable to consume any peptide in the given conditions, whereas it has been described elsewhere as the main peptide transporter for peptides longer than three amino acid residues in experiments in laboratory conditions. This controversy signifies the need in applied sciences for approaching experimental conditions to those prevalent in the industry for its more accurate characterization. Altogether, this work provides further evidence of the importance of peptides as a nitrogen source for yeast and their consequent positive impact on fermentation kinetics.