Thermonuclease is known as a specific virulence factor in Staphylococcus aureus. It is widely used as a genetic marker for detection of S. aureus in various types of food. Previous studies have revealed the existence of two functional thermostable nucleases encoded by two different genes (nuc1 and nuc2) in S. aureus. To identify the expression characteristics of these two genes, comparative mRNA analysis of nuc1 and nuc2 was carried out by quantitative real-time polymerase chain reaction (PCR). Distinct expression patterns were observed at different growth stages, and expression was under the control of the sae regulatory system in strain RN4220. The maximum level of nuc1 transcripts was at the post-exponential growth phase, and expression was notably down-regulated in a sae mutant. In contrast, nuc2 transcript levels declined after the early exponential phase, and they were slightly up-regulated in the sae mutant. Furthermore, unlike the expression of nuc1, which varied in three different S. aureus clinical strains, the transcription of nuc2 remained relatively constant. The nuc1 transcription level correlated well with thermonuclease activity results, which suggests that nuc1 plays a primary role in thermonuclease activity in S. aureus. This information will be useful for understanding thermonuclease gene function and alterations of regulation for pathogenesis and diagnosis of S. aureus.