Objective: The hypothalamic–pituitary–adrenal (HPA) axis has been proposed to be susceptible to fetal programming, the process by which an adverse fetal environment elicits permanent physiological and metabolic alterations predisposing to disease in later life. It is hypothesized that fetal exposure to poor circumstances alters the set point of the HPA axis, leading to increased HPA axis activity and subsequent increased cortisol concentrations. In this study, we tested the hypothesis that prenatal exposure to famine during different periods of gestation is associated with increased activity of the HPA axis. Design and methods: We assessed plasma cortisol concentrations after a dexamethasone suppression and an ACTH_1–24-stimulation test in a group of 98 men and women randomly sampled from the Dutch famine birth cohort. Cohort members were born as term singletons around the 1944–1945 Dutch famine. Results: Cortisol profiles after dexamethasone suppression and ACTH_1–24 stimulation were similar for participants exposed to famine during late, mid- or early gestation (P=0.78). Cortisol concentrations after dexamethasone suppression test did not differ between those exposed and those unexposed to famine in utero (mean difference −2% (95% confidence interval (CI) −27 to 23)). Neither peak cortisol concentration (20 nmol/l (95% CI −27 to 66)), cortisol increment (−5 nmol/l (95% CI −56 to 47)) or cortisol area under the curve post-ACTH_1–24 injection (4% (95% CI −4 to 12)) differed between exposed and unexposed participants. Conclusions: Prenatal famine exposure does not seem to affect HPA axis activity at adult age, at least not at the adrenal level. This does not exclude altered HPA axis activity at the levels of the hippocampus and hypothalamus.