The Gram-positive pathogen Streptococcus pyogenes injects a β-NAD + glycohydrolase (SPN) into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. In this compartment, SPN accelerates the death of the host cell by an unknown mechanism that may involve its β-NAD+-dependent enzyme activities. SPN has been reported to possess the unique characteristic of not only catalyzing hydrolysis of β-NAD+, but also carrying out ADP-ribosyl cyclase and ADP-ribosyltransferase activities, making SPN the only β-NAD+ glycohydrolase that can catalyze all of these reactions. With the long term goal of understanding how these activities may contribute to pathogenesis, we have further characterized the enzymatic activity of SPN using highly purified recombinant protein. Kinetic studies of the multiple activities of SPN revealed that SPN possessed only β-NAD+ hydrolytic activity and lacked detectable ADP-ribosyl cyclase and ADP-ribosyltransferase activities. Similarly, SPN was unable to catalyze cyclic ADPR hydrolysis, and could not catalyze methanolysis or transglycosidation. Kinetic analysis of product inhibition by recombinant SPN demonstrated an ordered uni-bi mechanism, with ADPribose being released as a second product. SPN was unaffected by product inhibition using nicotinamide, suggesting that this moiety contributes little to the binding energy of the substrate. Upon transformation, SPN was toxic to Saccharomyces cerevisiae, whereas a glycohydrolase-inactive SPN allowed for viability. Taken together, these data suggest that SPN functions exclusively as a strict β-NAD+ glycohydrolase during pathogenesis.