We developed a simple and sensitive enzyme-linked immunosorbent assay (ELISA) for human pulmonary surfactant protein D (SP-D). Human SP-D was purified from bronchoalveolar lavage fluids of patients with pulmonary alveolar proteinosis. Nine monoclonal antibodies (MAbs) were established from BALB/c mice immunized with the purified human SP-D. All MAbs were directed to either 43 kDa SP-D contained in lung lavage fluids of patients with pulmonary alveolar proteinosis or in amniotic fluids from healthy normal pregnancies. The ELISA is based on a sandwich method using two MAbs, 6B2 and 7C6. Cross-reactivity to human SP-A or rat SP-D was evaluated as below 0.6%. The recovery of different concentrations of SP-D ranged from 94.4% to 111.2%, and serial dilutions of amniotic fluids showed good linearity. SP-D concentrations in 21 amniotic fluids from normal pregnancies were measured by the ELISA. The mean concentration in amniotic fluids from pregnancies in the third trimester was significantly higher than that from earlier stages of gestation (p < 0.001), indicating that this ELISA may be applicable for prediction of fetal lung maturity.