The objectives of this study were to determine the extent of changes in concentrations of testosterone, growth hormone (GH) and insulin-like growth factor I (IGF-I) concentrations in blood plasma and to characterize the respective plasma-binding proteins of these two peptides during the onset of puberty in male calves. The jugular veins of six male Holstein calves (11-weeks-old) were catheterized and blood was collected every 3 days (one sample every 30 min for 8 h). Hormone concentrations in plasma were determined by specific radioimmunoassay. After incubation with [125I]IGF-I, IGF-I-binding proteins (IGFBPs) were separated by gel filtration; radioactivity was determined in each fraction. Western ligand blotting using radiolabelled hormones as ligand was also used to characterize the IGF-I- and GH-binding proteins in plasma. Puberty was characterized by a rapid (in 1 or 2 days) increase in mean concentrations of testosterone in plasma (from 0.5 to >2 ng ml−1) and a pulsatile release of the hormone. During puberty, IGF-I concentrations also increased rapidly in 8–10 days from ±50 ng ml−1 to > 150 ng ml−1, whereas concentrations of GH in plasma remained relatively stable during the experimental period. A significant correlation was observed between IGF-I and testosterone concentrations (r = 0.77; P < 0.001) throughout the experimental period. Three different IGF-I-binding protein fractions with apparent molecular masses of > 200, 150–170 and 45–65 kDa were found in plasma using gel filtration. The 150–170 kDa IGF-I-binding protein complex was more abundant after puberty, whereas the lower molecular mass fraction was slightly decreased. This release was confirmed by ligand blotting analysis. The binding subunit of the 150–170 kDa complex (molecular mass = 45–54 kDa; IGFBP-3) was more prominent after puberty, whereas the intensity of the 38 kDa band (IGFBP-2) was decreased. During this physiological step, migrant GH-binding proteins at 190, 58 and 31 kDa presented similar band intensities in all animals. In conclusion, puberty in male calves was associated with subsequent increases in amounts of testosterone, IGF-I and IGFBP-3 complex, whereas IGFBP-2 was reduced. IGF-I and perhaps IGFBPs could be used as indicators of the establishment of puberty. In practice, this factor could be better than testosterone for evaluating hormonal status because of its nycthemeral stability.