SummaryThe reports published thus far on prolactin and thyrotrophin secretion in patients with Klinefelter's syndrome are controversial. The aim of the present study was to investigate the interrelation between prolactin on one hand, and hormones of the hypothalamic‐pituitary‐gonadal axis and thyrotrophin, on the other, in males with Klinefelter's syndrome. Fifteen patients with Klinefelter's syndrome, aged between 17 and 43 years, and 15 healthy males, aged 22–35 years, were studied. Mean ± SD basal serum prolactin levels were 529.6±174.6 mU l‐1in the patients, and 270.1±113.0 mU l‐1in the control group (P<0.001). Following 200 μg thyrotrophin releasing hormone, an enhanced prolactin response was seen in the males with Klinefelter's syndrome. There was no evidence of any of the well‐known causes of hyperprolactinaemia. The response of thyrotrophin to thyrotrophin releasing hormone was more pronounced in Klinefelter patients in comparison with controls. Presumably, in Klinefelter's syndrome both alterations—of prolactin and thyrotrophin secretion—may be caused by decrease of testosterone levels or they could reflect a disturbance in neuroendocrine regulation with some neurotr