Abstract. The responses of biologically active LH (BIO-LH) and immunoreactive LH (RIA-LH) to acute stimulation with LRH (0.1 mg iv) were studied in 8 pubertal boys (9–15 years, 2nd–4th Tanner's stage), and in 10 healthy adult men (20–46 years). Serum levels of BIO-LH were assessed by an in vitro bioassay method based upon testosterone production by mechanically dispersed mouse Leydig cell preparations. In pubertal boys the mean BIO-LH/RIA-LH (B/I) ratio of basally secreted LH was significantly lower than in adult men (1.2 ± 0.2 (sem) and 2.2 ± 0.2 respectively, P < 0.01). After acute administration of LRH the mean B/I ratio of circulating LH showed a significant increase from the basal value in pubertal boys (2.6 ± 0.2, P < 0.01 vs basal values), whereas no significant difference in LH B/I ratios were demonstrated throughout the study period in adult men (2.1 ± 0.1, P = NS vs basal values). In agreement with this finding, the mean relative maximum response for BIO-LH (BIO-LH Δ%) was higher in pubertal boys than in adult men (1702.7 ± 500.3 and 499.6 ± 65.4% respectively, P < 0.05), whereas the mean RIA-LH Δ% was similar in both groups (609.1 ± 85.1 and 534.1 ± 75.5% respectively, P = NS). No significant differences were shown in the BIO-LH Δ area between pubertal boys (4.9 ± 0.9 area units × 103) and adult men (6.7 ± 1.2 area units × 103, P = NS), whereas the mean RIA-LH Δ area was significantly lower in the former group (1.9 ± 0.4 area units × 103 vs 3.2 ± 0.5 area units × 103, P < 0.05). Our study emphasizes that the pubertal pituitary possesses a greater responsiveness for BIO-LH than the adult pituitary, and that in pubertal boys acute stimulation with LRH evokes the release of a more bioactive form of LH.