Determinations of serum gonadotropin concentrations by ultra-sensitive methods have improved the diagnosis of pubertal disorders. The onset of puberty can be estimated by measuring serum gonadotropin pulsation, but as this requires serial nocturnal blood sampling, it is not a routine investigation. Gonadotropin measurements in first morning voided (FMV) urine samples could reflect the integrated nocturnal gonadotropin secretion and predict pubertal development earlier than daytime serum measurements. We studied the value of urinary LH (U-LH) measurements in FMV urine with reference to serum LH (S-LH) levels using an ultrasensitive time-resolved immunofluorometric assay in samples from 297 children and adolescents (145 boys and 152 girls, aged 5-15 yr) with known pubertal stages (Tanner 1-5). Stage 1 subjects (prepubertal) were divided into 5 age groups to assess whether there is an increase in LH before clinical signs of puberty can be detected. The correlation between FMV urine and S-LH values was good (r = 0.64; P <0.0001). The 2 oldest groups of prepubertal subjects (11 and 12 yr) had significantly higher (P <0.001) U-LH concentrations than the 3 younger groups. This difference was less marked for S-LH. A significant increase in FMV U-LH concentration occurs before the first clinical signs of puberty in a sex-independent fashion. Our data indicate that FMV U-LH measurement is a clinically relevant, noninvasive method for the evaluation of pubertal development, and it may be helpful in the investigation of pubertal disorders.