Nicotinamide deamidase (YNDase) has been purified from yeast through the use of a six-step procedure that includes molecular-sieve high performance liquid chromatography. The final preparation was homogeneous by the criteria of sodium dodecyl sulfate-gel electrophoresis, and the enzyme specific activity was determined to be 175 mumol of nicotinate formed per min/mg enzyme. Gel electrophoresis and molecular-sieve high performance liquid chromatography were employed also to characterize YNDase as a monomeric protein with a molecular weight of 34,000. A Km value for nicotinamide of 33 microM was determined for the deamidase activity at pH 6, and a pH range for optimal stability of 6-8.5 was established for this enzyme. The YNDase activity was also examined over a pH range at several substrate concentrations and both the log Vmax and log Vmax/Km plots versus pH suggested that a protonated amino acid residue with an apparent pKb value of 7.8 was essential to this activity. During an in vitro assay of the YNDase-catalyzed formation of nicotinate, ammonia was generated and detected chemically. Inhibition of the YNDase activity by nicotinaldehyde suggested the presence of either an essential lysine (Schiff's base formation) or cysteine residue (thiohemiacetal intermediate) at the YNDase active site. The relatively large value of the nicotinaldehyde inhibition constant (Ki = 68 microM), the observation that this analogue is a noncompetitive inhibitor of nicotinate formation, and the fact that this inhibition can be rendered irreversible through incubation with sodium borohydride, indicates that a Schiff's base intermediate is more likely to occur upon incubation of YNDase with nicotinaldehyde. However, YNDase is inactivated completely and irreversibly by N-ethylmaleimide at pH 6, and the enzyme is protected against this modification by either nicotinamide or nicotinate. These results suggest that both nicotinate and nicotinamide bind to YNDase, even though the enzymatic reaction is essentially irreversible, and that a cysteine residue may be present at the YNDase active site.