We developed dioxatane-based immunochemiluminometric assays (ICMAs) for lutropin (LH) and follitropin (FSH), using monoclonal antibodies. These ICMAs have a minimal detectable dose (analytical sensitivity) of 0.01 IU/L, extending the lower limit of sensitivity 10-fold (from 0.10 IU/L) when compared with immunoradiometric assays (IRMA) (second generation), and thus provide a true third-generation assay. Daytime FSH and LH concentrations were measured in 236 boys and 195 girls. Unlike the previous assays, all the samples had detectable concentrations of LH and FSH. In agreement with results from earlier methods, the present results indicate that for both sexes mean FSH and LH concentrations are relatively high during the early months of life, fall to baseline prepubertal concentrations by 12-18 months, and remain low until the onset of puberty. During puberty, the mean concentrations of FSH and LH increase significantly in both girls and boys with each stage of puberty, but there is considerable overlap between stages. These third-generation FSH and LH ICMAs reliably separate daytime plasma FSH and LH concentrations of prepubertal children from those of sexually mature children, and therefore can more reliably distinguish between the major causes of precocious puberty (e.g., gonadotropin dependent and independent). Our LH assay is also useful in monitoring the gonadotropin-releasing hormone therapy of patients with gonadotropin-dependent precocious puberty.