The efficacy of testosterone undecanoate (TU) treatment in constitutional delay of growth (CHD) is well recognized. We investigated its role in initiating puberty. Sera taken prior to, just after 6 months on and after 6 months off treatment with TU (20 mg daily) were analyzed from eight boys and compared to results from eight boys receiving placebo. Prostate specific antigen (PSA) and sex hormone binding globulin (SHBG), sleep-entrained pulsatility and mean overnight luteinizing hormone (mLH), and morning testosterone (T) levels were measured. Free androgen index (FAI) was calculated. Testicular volume (TV) and growth parameters were assessed. During treatment, there was a significant increase in height velocity in boys taking TU vs placebo (mean +/- SD: 5.7 +/- 2.0 vs 3.2 +/- 0.9 cm/year, p = 0.008) but no significant differences were observed in regard to LH pulsatility, mLH, T, SHBG, FAI, PSA and TV values. PSA was detectable in four patients (two each in the TU and placebo groups) at 6 months off treatment indicating pubertal progression. Among the hormones measured, only pretreatment mLH levels were significantly higher in the PSA-positive patients compared to 12 PSA-negative patients (mean +/- SEM: 1.5 +/- 0.39 vs 0.37 +/- 0.06 IU/l, p < 0.001). In conclusion, TU treatment shows no significant effect on initiation or advancement of puberty despite its resultant growth acceleration. Among the hormonal changes studied, mLH levels were the earliest indicator of pubertal initiation.