Felinine is a branched-chain sulfur amino acid present in the urine of certain Felidae, including domestic cats. The objective of the present study was to determine if additional cystine and/or dietary N would increase felinine and N-acetylfelinine excretion by intact male cats fed a low-protein (LP) diet. Feeding five adult intact male cats an LP diet (18·8 % of metabolisable energy (ME) as protein) v. a high-protein diet (38·6 % of ME as protein) resulted in a trend (P = 0·08) for decreased urinary felinine and no change in N-acetylfelinine excretion. In a 23 d study, when the LP diet was supplemented with l-cystine at 9·3 g/kg DM, urinary felinine:creatinine ratio showed a linear two-fold (121 %) increase (P < 0·01) from 0·24 (sem 0·05) to 0·53 (sem 0·13) after 10 d. Subsequent feeding of the LP diet resulted in a decrease in felinine excretion to base levels. Plasma γ-glutamylfelinylglycine concentrations were consistent with the excretion of felinine. Supplementation of the LP diet with l-cystine (9·3 g/kg DM), dispensable amino acids and arginine to a second group (n 5) also resulted in a significant (P < 0·01) but smaller (+72 %) increase in the daily felinine:creatinine ratio (0·25 (sem 0·04) to 0·43 (sem 0·05)). The degree of felinine N-acetylation within groups was unaffected by dietary addition and withdrawal of amino acids. The results indicate that felinine synthesis is regulated by cystine availability, and that arginine may be physiologically important in decreasing felinine biosynthesis in intact male cats.