A sensitive nonisotopic immunoassay for the determination of 17-hydroxyprogesterone (17-OHP) levels in saliva was developed. The new time-resolved fluorometric immunoassay employs a specific polyclonal anti-17-OHP antiserum immobilized onto microtiter plates, a 17-OHP-biotin conjugate as a tracer, and streptavidin-europium a as secondary probe. The lower detection limit of the assay is 23.6 pmol/L (mean -3 s of a 22-fold zero determination) corresponding to 0.39 pg/well. The coefficients of intraassay variation are 8.8, 5.3, and 8.3% at the respective concentrations of 90.9, 454.5, and 1363.5 pmol/L. The coefficients of interassay variation are 8.8, 5.3, and 8.3% at the respective concentrations. Saliva was collected in commercially available devices. Reference ranges were established using 394 saliva samples from 132 healthy children, adolescents, and adults. Morning, midday, and evening levels of 17-OHP levels in saliva varied significantly in all age groups with morning levels being higher than midday and evening levels. Saliva samples (n = 57) were also obtained from 18 children with congenital adrenal hyperplasia (CAH). Salivary 17-OHP levels in the limited number of CAH patients studied ranged from 121 to 106 050 pmol/L. In conclusion 1) a new, sensitive nonisotopic immunoassay for measurement of 17-OHP in saliva has been developed; 2) reference ranges for healthy children, adolescents, and adults have been established; 3) there is a circadian pattern of 17-OHP levels in saliva at all ages; and 4) measurement of 17-OHP in saliva should be further evaluated over a longer period of time as a potentially reliable and powerful technique to monitor metabolic control in patients with CAH. As 17-OHP levels in saliva are stable for > 10 wk at 4°C, the technique is ideally suited for outpatient sampling.