A simple, rapid, sensitive, and reliable ELISA test for the detection of urinary human chorionic gonadotropin (hCG) has been developed using penicillinase (EC no. 3.5.2.6) as the label and a microtiter ELISA plate as a solid support for antibody. The results of the test is assessed visually for the dark blue that indicates the presence of hCG. A minimum of 500 mU of hCG/ml of urine can be detected easily and the test can be completed in 2 hours with good precision. Accurate negative results are obtained in urine samples of normally ovulatory women, postmenopausal women, and adult males, suggesting that the physiologic concentrations of luteinizing hormone do not influence the assay results. In samples referred for pregnancy detection 95.5% accurate results were obtained when compared with histopathologic and/or clinical findings. There were 2 false-negative results, and only in 4 of 132 subjects studied were false-positive results obtained. This test, if properly performed, should prove a valuable tool in the detection of hCG as early as 30 to 35 days from the last menstrual period.