A number of immunological functions are dependent on circadian rhythms and regular sleep. This has impact on the type and magnitude of immune responses following antigenic challenge, for example in vaccination. Little is known about the underlying mechanisms. One possibility may be the circadian and sleep-dependent modulation of CD4+CD25- T cell responses by CD4+CD25+ natural regulatory T cells (nTreg). In a variety of studies, nTreg have been shown to regulate T cell responses negatively. Thus, we investigated the influence of sleep and circadian rhythm on the number and function of nTreg as well as on the function of CD4+CD25- T cells. Seven healthy young men were examined under defined conditions on two occasions, i.e. during sleep and sleep deprivation. Venous blood was drawn periodically; numbers of nTreg, suppressive activity of nTreg, interleukin-2 production and proliferation of CD4+CD25- T cells were explored in vitro. nTreg counts revealed a significant circadian rhythm with highest levels during the night (mean 95 nTreg/µl) and lowest levels during the day (mean 55 nTreg/µl). During normal sleep, the suppressive activity of nTreg was highest at 02.00 h and somewhat lower at 15.00 h. Surprisingly, almost no suppressive activity was present at 07.00 h. Deprivation of sleep abrogated this rhythm. CD4+CD25– T cell proliferation was dampened significantly by sleep deprivation. This is the first study in human cells to show that nTreg number and function follow a rhythm across the 24-h period. Furthermore, sleep deprivation severely disturbs the functional rhythm of nTreg and CD4+CD25– T cells.