Experimental studies in animals show that prenatal undernutrition leads to lifelong alterations in the activity of the hypothalamic-pituitary-adrenal (HPA) axis. Some studies have shown associations between low birth weight and an increased HPA response to psychological stress. We tested the hypothesis that prenatal exposure to the Dutch 1944-1945 famine leads to an elevated HPA response to psychological stress in adult life. We measured salivary cortisol responses to a psychological stress protocol among 694 adults who were born as term singletons in Amsterdam, the Netherlands, around the time of the 1944-1945 Dutch famine. We compared cortisol profiles of participants exposed to famine during late (n=120), mid (n=100), or early gestation (n=62) to profiles of participants unexposed to famine during gestation (n=412). The mean increase in cortisol concentrations from baseline was 30% (95% CI 23-37). There were no statistically significant differences in the mean profile of cortisol response to the psychological stress protocol between participants exposed and unexposed to famine in utero. The mean sex and BMI adjusted difference in cortisol response for those exposed compared to those unexposed was -6% (95% CI: -15 to 2). The cortisol profiles of those exposed in late (-4% [95% CI: -16 to 7]), mid (-9% [95% CI: -22 to 3]) or early gestation (-4% [95% CI: -20 to 10]) did not differ from the profile of those unexposed to famine. We conclude that prenatal exposure to famine does not seem to be associated with the response of the HPA axis to psychological stress. However, the stress protocol we have used may have been unsuccessful in inducing a strong enough HPA axis activation to be able to detect famine related differences.