The aim of our study was to examine the relationship between bone mineral density (BMD) and serum ghrelin, insulin-like growth factor-1 (IGF-1), IGF-binding protein 3 (IGFBP-3), and testosterone levels in boys at different stages of puberty. The study included 60 healthy nonobese Estonian schoolboys at the age of 10-18 years. Subjects were divided in three groups (20 boys in each) based on the results of self-assessment using illustrated questionnaire of pubertal stage (G1, I; G2-G3, II; G3-G4, III). Morning fasting blood samples were collected for analysis of ghrelin, testosterone, IGF-1, and IGFBP-3. Total body BMD, lumbar BMD, lumbar apparent volumetric BMD (BMAD), and bone mineral content (BMC) were measured by DXA. Serum testosterone concentration was the most important biochemical predictor of BMD in the total group, explaining 48.8% of variability in total body BMD, 51.4% in lumbar BMD, and 36.8% in lumbar BMAD. Body mass and height were both related to BMD and BMC throughout puberty. The serum IGF-1/IGFBP-3 ratio was correlated with serum testosterone (r = 0.69) and ghrelin (r = -0.58) levels, but also with total BMD (r = 0.39), lumbar BMD (r = 0.42; P < 0.001 in all cases), BMAD (r = 0.29; P < 0.01), and total BMC (r = 0.48; P < 0.001). We conclude that serum testosterone concentration and serum IGF-1/IGFBP-3 molar ratio are the major determinants of bone mineral density in boys at different pubertal stages. Serum ghrelin concentration did not appear to have a direct independent effect on BMD. If present, the association may be mediated through sex hormones and the GH-IGF-I axis.