The physiological importance of endogenous ghrelin in the regulation of growth hormone (GH) secretion is still unknown. To investigate the regulation of ghrelin secretion and pulsatility, we performed overnight ghrelin and growth hormone sampling every 20 minutes for 12 hours in 8 healthy male subjects [age 37 ± 5 (mean±SD) years old, BMI 27.2 ±2.9 kg/m²]. Simultaneous growth hormone and ghrelin levels were assessed to determine the relatedness and synchronicity between these two hormones in the fasted state, during the overnight period of maximal endogenous growth hormone secretion. Pulsatility analyses were performed to determine simultaneous hormonal dynamics and investigate the relationship between GH and ghrelin using cross-correlation and Cross-ApEn (X-ApEn) analyses. Subjects demonstrated 3.0±2.1 ghrelin pulses/12 hours and 3.3±0.9 growth hormone pulses/12 hours. The mean normalized ghrelin entropy (ApEn) was 0.93±0.09 indicating regularity in ghrelin hormone secretion. The mean normalized X-ApEn was significant between ghrelin and growth hormone (0.89±0.12), demonstrating regularity in co-secretion. In addition, we investigated the ghrelin response to standard GH secretagogues [growth hormone releasing hormone (GHRH) alone and combined GHRH-arginine], in separate testing sequences separated by 1 week. Our data demonstrate that in contrast to GHRH alone, which had little effect on ghrelin, combined GHRH and arginine significantly stimulated ghrelin with a maximal peak at 120 minutes, representing a change of 66 ± 14 pg/mL (P=0.001 by repeated measures ANOVA and P=0.02 for GHRH vs. combined GHRHarginine by MANOVA). We demonstrate relatedness between ghrelin and GH pulsatility, suggesting either that ghrelin participates in the pulsatile regulation of GH or that the two hormones are simultaneously co-regulated, e.g. by somatostatin or other stimuli. Furthermore, the differential effects of GHRH alone vs. GHRH-arginine suggest that inhibition of somatostatin tone may increase ghrelin. These data provide further evidence of the physiologic regulation of ghrelin in relationship to growth hormone.