We have recently described a competitive binding assay for rat insulin-like growth factor-binding protein-3 (IGFBP-3) based on the ability of IGFBP-3 to form a ternary complex with the acid-labile subunit (ALS) in the presence of IGF-I. Using this assay we studied groups of male (n = 6) and female rats (n = 6) at 20, 30, 40, 50, 60, 80, and 130 days of age. Nonfasting serum levels of IGFBP-3 were compared with those of total (extractable) IGF-I (tIGF-I) and ALS as well as IGFBP-3 determined by ligand blotting. Additionally, we studied the relationship between ultrafiltered free IGF-I (fIGF-I) and immunoassayable IGFBP-1. IGFBP-3 was dependent on age only (P < 0.0001), but tended to be higher in males than in females (P = 0.06); between 20-130 days levels increased from 6.5 +/- 1.7 to 73.6 +/- 7.2 nmol/liter in males and from 5.4 +/- 1.6 to 51.3 +/- 8.0 nmol/liter in females. IGFBP-3 correlated positively with tIGF-I (r = 0.90; P < 0.0001), ALS (r = 0.92; P < 0.0001), and IGFBP-3, as determined by ligand blotting (r = 0.88; P < 0.0001). The molar ratio of IGFBP-3 to tIGF-I increased from 0.23 +/- 0.04 to 0.76 +/- 0.04 (P < 0.0001) without any sex dependence. An age- and sex-dependent decrease in IGFBP-1 was observed (P < 0.0001), from 10.9 +/- 2.5 to 1.2 +/- 0.2 nmol/liter in females and from 8.9 +/- 0.7 to 0.2 +/- 0.04 nmol/liter in males. Free IGF-I (fIGF-I) increased with age (from 0.7 +/- 0.2 to 7.1 +/- 0.5 nmol/liter; P < 0.0001), and levels were inversely correlated with IGFBP-1 (r = -0.80; P < 0.0001). In young rats, IGFBP-1 circulated in a 10-fold molar excess over the level of fIGF-I, whereas in older rats, fIGF-I exceeded IGFBP-1 by an average of 9-fold in females and by up to almost 60-fold in males. We conclude that in rats 1) IGFBP-3 and fIGF-I are strongly age dependent; 2) IGFBP-3 correlates positively with ALS and tIGF-I; and 3) fIGF-I and IGFBP-1 are inversely correlated. This is in accordance with clinical findings. However, in humans the adult level of fIGF-I rarely exceeds 0.3 nmol/liter, and IGFBP-1 usually circulates in excess of fIGF-I. Thus, our results also imply species differences in the IGF systems of humans and rats.