Time resolved immunofluorometric assays (IFMAs) for determining luteinizing hormone (LH) and follicle stimulating hormone (FSH) were developed, based on commercial IFMA kits designed for determining LH and FSH. Both kits used capture antibodies to bind to the LH or FSH structure, therefore avoiding the use of radioisotopes required for conventional radioimmunoassays. The kit manufacturer's procedure for determining LH was modified by using a robotic sample processor to pipette urine samples onto a single plate, reducing the pipetting time, adding the assay buffer before the sample, and increasing the recommended incubation time from 15 to 60 minutes. The recommended procedure for determining FSH was modified by using the robotic sampler to pipette samples onto the assay plates and adding the assay buffer before the sample. Calibration plots were linear over the range 0.25 to 256 milliinternational units per milliliter (mIU/ml) FSH and 0.6 to 250mIU/ml LH. In recovery experiments in which urine samples were spiked with LH or FSH gonadotropin at concentrations of 1.61 to 127mIU/ml, recoveries of 98.1 and 96.4%, respectively, were obtained. The means for between and within assay precision, measured by the coefficients of variation, were 5.2 and 4.8% for FSH and 6.7 and 6.3% for LH, respectively. The procedures were applied to determining the concentrations of FSH and LH in urine samples collected over 19 menstrual cycles from ten healthy, nonpregnant women. Thirteen menstrual cycles were characterized as normal and six as atypical. The LH and FSH data were consistent with normal ovulatory processes occurring, although occasional bimodal surges in LH excretion were observed. The authors conclude that the nonradioisotopic IFMA assays offer sensitive, convenient, and noninvasive methods for determining urinary FSH and LH for epidemiological and clinical studies.