The globally dominant, invasive M1T1 strain of group A Streptococcus (GAS) harbors polymorphisms in the promoter region of an operon that contains the genes encoding streptolysin O (SLO) and NAD-glycohydrolase (NADase), which results in high-level expression of these toxins. While both toxins have been shown experimentally to contribute to pathogenesis, many GAS isolates lack detectable NADase activity. Reduced or absent enzymatic activity can be associated with a variety of point mutations in nga, the gene encoding NADase. However, nga has not been observed to contain early termination codons or mutations that would result in a truncated protein, even when the gene product contains missense mutations that abrogate enzymatic activity. We found that expression of NADase, either enzymatically active or inactive, is associated with increased abundance of SLO in culture supernatants and increased SLO-mediated toxicity for keratinocytes. Conversely, production of SLO protects NADase from proteolytic cleavage. Analytical gel filtration chromatography demonstrated physical association of purified SLO and NADase in solution, a result confirmed by biolayer interferometry. After glutaraldehyde treatment of a mixture of the two proteins, a crosslinked species of 110.7 kD could be detected by mass spectrometry, consistent with a heterodimer of 1:1 binding stoichiometry. Thus, SLO and NADase interact in solution, and both the translocation and catalytic domains of NADase are required for maximal binding between the two toxins. We conclude that expression of full-length NADase is under positive selection because binding of NADase to SLO stabilizes both toxins, thereby enhancing GAS survival in the human host.