Using sandwich enzyme-linked immunosorbent assay (ELISA), the production of staphylococcal enterotoxin (SE) H was determined in 22 Staphylococcus aureus isolates bearing the seh gene. Samples of supernatants were taken at four time points corresponding to exponential phase (optical density at 600 nm [OD(600)] 0.3 to 0.6), late exponential phase (OD(600) 2 to 4), early stationary phase (OD(600) 4 to 6), and late stationary phase (OD(600) 7 to 12). In four isolates, SEH was detectable at a very low level at the first time point. In 18 isolates, the earliest SEH production was detected in the late exponential phase. For all isolates, there was an increase of SEH concentration with time. Western blot analysis revealed that SEH production, similar to SEA, started in the early exponential phase (OD(600) ∼ 0.5). Isolates with high SEH productivity, as measured by ELISA, demonstrated a higher seh transcription as well. sec transcription was induced in the stationary phase. An induction in the sea transcript was observed during mid- to late exponential phase. Expression profile of seh was similar to that of sea. We showed that the seh expression profile is similar to that of Agr-independent sea and not to that of Agr-dependent sec genes. SEH can be effectively expressed at low bacterial counts, meaning that even in an environment not favorable for S. aureus growth, seh-bearing strains can pose a risk for food safety.