A rapid and sensitive method has been developed to detect a-fetoprotein (AFP) in human serum by a two-site sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies (MAbs) for human AFP within 1 h. To obtain the most sensitive and reliable MAbs. 12 kinds of MAbs (HPJ1 to HPJ12) as a capture antibody and 4 kinds of horseradish peroxidase (HRP) conjugated MAbs as a tracer antibody were investigated. Among these, only HPJ 10-HRP conjugated HPJ 1 (HPJ 10-HPJ $1^*$) and HPJ 11-HRP conjugated HPJ 10 (HPJ 11-HPJ $10^*$) were chosen as candidates based on the linearity of the standard curve and the sensitivity of the assay. To further characterize these two pairs. MAbs against human AFP were purified from hybridoma cells. conjugated with HRP. and then characterized to optimize the two-site sandwich ELISA The HPJ 10-HPJ $1^*$ pair showed a sensitivity of 1 ng/ml and a better reproducibility than the HPJ 11-HPJ $10^*$ pair when the human sera were incubated at $37^{circ}C$ for 30 min. The results obtained for 480 randomly selected human sera showed 0~20 ng/ml of AFP values for the normal human sera. To test the utility of our kit, AFP concentrations were determined for 951 human sera (including 85 normal sera, 480 random blood sera, 213 HBsAg-positives. 50 anti-HCV antibody positives. and 47 malignant diseases) and compared with other commercially available AFP detecting kits. These results show that the present two-site sandwich ELISA method is a rapid, sensitive, and reliable procedure for detecting AFP in human serum.