Total Protein S (tPS) and free Protein S (fPS) antigen levels were measured in 3788 healthy blood donors. Men had higher levels of both parameters than women (P < 0.001). Age had no effect on tPS in men, although there was a slight reduction in fPS levels with increasing age. In women increasing age was associated with a significant increase in tPS levels (P < 0.001) but had no effect on fPS after adjustment for menopausal state. Oral contraceptive pill (OCP) use significantly lowered tPS but had no effect on fPS. In post-menopausal women, hormone replacement therapy (HRT) use had no statistically significant effect on either tPS or fPS. Donors with tPS or fPS levels in the lowest percentile (n = 56) were retested; only nine with repeat low levels were identified, eight of whom had persistently low levels over a 4-7-year follow-up. Acquired deficiency was excluded. When possible, family studies were performed, leading to an estimate of prevalence of familial PS deficiency of between 0.03% and 0.13% in the general population.