Proteolysis during cheese fermentation and ripening can lead to the formation of peptides with various biological activities including antihypertensive, antioxidant and immunomodulatory properties. Factors that affect proteolysis may also affect the concentration of bioactive peptides and the consequent bioactivity of ripened cheeses. This study evaluated the effect of salt content on the relative intensity of the antihypertensive peptide αs1-CN (f1-9). Cheeses were produced according to the conventional technology and divided into three batches which were submitted to different brining times, thus obtaining control cheese (1.68% salt) and cheeses with 25% and 50% salt reduction. Analysis of pH 4.6 and 70% ethanol soluble peptides was carried out by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) after 1, 15, 30, 45, and 60 days of ripening. The effect of salt reduction on the relative intensity of the peptide αs1-CN (f1-9) (m/z 1441) was evaluated by analysis of variance (ANOVA) and Tukey test for comparison of means at a significance level of 5%. Cheeses with a reduction of 25 and 50% in the salt content presented lower relative intensity of the peptide αs1-CN (f1-9) (p=0.0008) when compared to the control cheese. Possibly, the higher salt content inhibited the microbial and enzymatic activities, resulting in lower hydrolysis and consequent accumulation of this peptide during the ripening of the control cheese. Although salt reduction is nutritionally desirable, it may negatively affect the concentration of a peptide with antihypertensive activity. Additional technological resources for the manufacture of Prato cheese with reduced salt content and high concentration of antihypertensive peptides should be explored.