The effect of settling on mass balance and biodegradation characteristics of domestic wastewater and on denitrification potential was studied primarily using model calibration and evaluation of oxygen uptake rate profiles. Raw domestic wastewater was settled for a period of 30 minutes and a period of 2 hours to assess the effect of primary settling on wastewater characterization and composition. Mass balances in the system were made to evaluate the effect of primary settling on major parameters. Primary settling of the selected raw wastewater for 2 hours resulted in the removal of 32% chemical oxygen demand (COD), 9% total Kjeldahl nitrogen, 9% total phosphorus, and 47% total suspended solids. Respirometric analysis identified COD removed by settling as a new COD fraction, namely settleable slowly biodegradable COD (X(ss)), characterized by a hydrolysis rate of 1.0 day(-1) and a hydrolysis half-saturation coefficient of 0.08. A model simulation to test the fate and availability of suspended (X(s)) and settleable (X(ss)) COD fractions as carbon sources for denitrification showed that both particulate COD components were effectively removed aerobically at sludge ages higher than 1.5 to 2.0 days. Under anoxic conditions, the biodegradation of both COD fractions was reduced, especially below an anoxic sludge retention time of 3.0 days. Consequently, modeling results revealed that the settleable COD removed by primary settling could represent up to approximately 40% of the total denitrification potential of the system, depending on the specific configuration selected for the nitrogen removal process. This way, the results showed the significant effect of primary settling on denitrification, indicating that the settleable COD fraction could contribute an additional carbon source in systems where the denitrification potential associated with the influent becomes rate-limiting for the denitrification efficiency.