There are data indicating that cholinergic activity is precociously impaired in Down’s syndrome (DS). On the other hand, acetylcholine as well as arginine (ARG) play a major stimulatory role in the neural control of growth hormone (GH) secretion in humans, likely acting via the inhibition of hypothalamic somatostatin release. The aim of the present study was to verify the effects of pyridostigmine (PD, 120 mg p.o.), a cholinesterase inhibitor, and ARG (0.5 g/kg i.v.) on the growth hormone-releasing hormone (GHRH) (1 µg/kg i.v.)-induced GH rise in 15 adult patients with DS (M/F: 8/7; age 26.5 ± 2.2 years; body mass index, BMI: 25.7 ± 1.0 kg/m²) in which the potentiating effect of PD on GH secretion has been reported to be reduced. The results in DS were compared to those in 15 normal subjects (NS) (M/F: 8/7; age: 30.0 ± 1.3 years; BMI: 21.4 ± 0.4 kg/m²). Basal GH and insulin growth factor I (IGF-1) levels in DS (1.8 ± 0.7 and 206.5 ± 21.0 µg/l) were similar to those in NS (1.4 ± 0.3 and 179.4 ± 11.0 µg/l). The GH response to GHRH alone in DS (526.5 ± 120.1 µg/l/h) was lower (p < 0.05) than that recorded in NS (895.4 ± 153.7 µg/l/h). The GHRH-induced GH rise was potentiated by PD both in DS (1,138 ± 184.2 µg/l/h; p < 0.02 vs. GHRH alone) and in NS (2,213.8 ± 212.8 µg/l/h; p < 0.005 vs. GHRH alone); however, as the percent potentiating effect of PD was similar in both groups (215 and 247%, respectively) the GH response to GHRH+PD in DS was lower (p < 0.005) than that in NS. The GHRH-induced GH rise was also potentiated by ARG in both DS (2,243 ± 362.4 µg/h; p < 0.001 vs. GHRH alone) and NS (2,764.3 ± 325.7 µg/l/h; p < 0.005 vs. GHRH alone). As the percent potentiating effect of ARG in DS was more marked than in NS (425 vs. 308%, respectively), the GH response to GHRH+ARG became similar in both groups. No sex-related difference was found in the GH response to various stimuli both in DS and NS. In conclusion, these data demonstrate that the potentiating effect of PD but not that of ARG is impaired in adults with DS in whom a reduced somatotrope responsiveness to GHRH is present. These findings indicate that in DS the pituitary GH releasable pool is fully preserved while an impairment of the tuberoinfundibular cholinergic pathways could lead to somatostatinergic hyperactivity and low somatotrope responsiveness to GHRH.