Protein hydrolysis and amino acid metabolism contribute to the beneficial effects of sourdough fermentation on bread quality. In this work, genes of Lactobacillus sanfranciscensis strain DSM 20451 involved in peptide uptake and hydrolysis were identified and their expression during growth in sourdough was determined. Screening of the L. sanfranciscensis genome with degenerate primers targeting prt and analysis of proteolytic activity in vitro provided no indication for proteolytic activity. Proteolysis in aseptic doughs and sourdoughs fermented with L. sanfranciscensis was inhibited upon the addition of an aspartic protease inhibitor. These results indicate that proteolysis was not linked to the presence of L. sanfranciscensis DSM 20451 and that this strain does not harbor a proteinase. Genes encoding the peptide transport systems Opp and DtpT and the intracellular peptidases PepT, PepR, PepC, PepN, and PepX were identified. Both peptide uptake systems and the genes pepN, pepX, pepC, and pepT were expressed by L. sanfranciscensis growing exponentially in sourdough, whereas pepX was not transcribed. The regulation of the expression of Opp, DtpT, and PepT during growth of L. sanfranciscensis in sourdough was investigated. Expression of Opp and DtpT was reduced approximately 17-fold when the peptide supply in dough was increased. The expression of PepT was dependent on the peptide supply to a lesser extent. Thus, the accumulation of amino nitrogen by L. sanfranciscensis in dough is attributable to peptide hydrolysis rather than proteolysis and amino acid metabolism by L. sanfranciscensis during growth in sourdough is limited by the peptide availability.