The production of hydrogen sulfide from stored liquid swine manure as influenced by the manure settling characteristics and the initial sulfate concentration was studied in the laboratory. The manure was settled in a 30-cm diameter ×120-cm high column for 24 h and then divided into three layers based on the solids content. The manure from different layers were placed in separate closed jars and stored for 30 days at 22 ± 2°C. The total sulfide, total solids, volatile solids, pH in the manure, and the hydrogen sulfide in the effluent gas stream were measured. The sulfide production rate was highest for all three manure layers during the first 5-10 days of storage. The top manure layer had the lowest solids content and the highest sulfide concentration. However, because of the lower pH levels in the bottom layer, the concentration of molecular H2S is higher in the bottom layer compared with the top layer. Mathematical equations for predicting hydrogen sulfide concentration and production rates in the different manure layers were derived. Three levels of initial sulfate concentration (5.89, 110, and 275 mg/L as SO4 2–) were used to test the effect of initial sulfate concentration in the liquid manure on sulfide production. Higher initial sulfate concentration in the manure resulted in higher sulfide concentration during the storage period. From the observations made, it was confirmed that reducing the sulfate concentration in the water supply would help reduce the sulfide production in the pit.