PA subunit of influenza RNA-dependent RNA polymerase deserves constantly increasing attention due to its essential role in influenza life cycle. N-terminal domain of PA (PA-Nter) harbors endonuclease activity, which is indispensable in viral transcription and replication. Interestingly, existing literature reports on in vitro ion preferences of the enzyme are contradictory. Some show PA-Nter activity exclusively with Mn2+, whereas others report Mg2+ as a natural cofactor. To clarify it, we performed a series of experiments with varied ion concentrations and substrate type. We observed cleavage in the presence of both ions, with a slight preference for manganese, however PA-Nter activity highly depended on the amount of residual, co-purified ions. Furthermore, to quantify cleavage reaction rate, we applied fluorescence cross-correlation spectroscopy (FCCS), providing highly sensitive and real-time monitoring of single molecules. Using nanomolar ssDNA in the regime of enzyme excess, we estimated the maximum reaction rate at 0.81± 0.38 and 1.38± 0.34 nM/min for Mg2+ and Mn2+, respectively. However, our calculations of PA-Nter ion occupancy, based on thermodynamic data, suggest Mg2+ to be a canonical metal in PA-Nter processing of RNA in vivo. Presented studies constitute a step toward better understanding of PA-Nter ion-dependent activity, which will possibly contribute to new successful inhibitor design in the future.