Background: In adults with Prader-Willi syndrome (PWS), limited information is available about pituitary function, more specifically the prevalence of growth hormone deficiency (GHD). The aim of this study was to gain more insight into endocrine function in PWS adults, with emphasis on GH secretion. Methods: 15 randomly selected adult PWS individuals were included and 14 healthy brothers and sisters served as a control group. Main outcome measures were IGF-I, IGFBP-3 and peak GH level after a combined GHRH-arginine test. Other pituitary hormone deficits are diagnosed based on serum levels of the concerning hormones. The size of the pituitary gland was measured on MRI images. Results: In PWS adults, IGF-I levels were low and IGFBP-3 levels normal when compared to healthy controls. GHD was diagnosed in 8–38% of the PWS patients, depending on the criteria used. Hypogonadism was present in 87% of the patients. Hypothyroidism and adrenal insufficiency could also be demonstrated. Anterior pituitary size was lower in PWS individuals when compared to healthy controls. Conclusion: In this study, pituitary hormone deficiencies are demonstrated in a considerable number of adults with PWS, hypogonadism and GHD being most prominent. Furthermore, the anterior pituitary is smaller in comparison with healthy controls.