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Flood pulse inundation of riparian forests alters rates of nutrient retention and organic matter processing in the aquatic ecosystems formed in the forest interior. Along the Middle Rio Grande (New Mexico, USA), impoundment and levee construction have created riparian forests that differ in their inter-flood intervals (IFIs) because some floodplains are still regularly inundated by the flood pulse (i.e., connected), while other floodplains remain isolated from flooding (i.e., disconnected). This research investigates how ecosystem responses to the flood pulse relate to forest IFI by quantifying nutrient and organic matter dynamics in the Rio Grande floodplain during three years of experimental flooding of the disconnected floodplain and during a single year of natural flooding of the connected floodplain. Surface and subsurface conditions in paired sites (control, flood) established in the two floodplain types were monitored to address metabolic and biogeochemical responses. Compared to dry controls, rates of respiration in the flooded sites increased by up to three orders of magnitude during the flood pulse. In the disconnected forest, month-long experimental floods produced widespread anoxia of four-week duration during each of the three years of flooding. In contrast, water in the connected floodplain remained well oxygenated (3–8 ppm). Material budgets for experimental floods showed the disconnected floodplain to be a sink for inorganic nitrogen and suspended solids, but a potential source of dissolved organic carbon (DOC). Compared to the main stem of the Rio Grande, floodwater on the connected floodplain contained less nitrate, but comparable concentrations of DOC, phosphate-phosphorus, and ammonium-nitrogen. Results suggest that floodplain IFI drives metabolic and biogeochemical responses during the flood pulse. Impoundment and fragmentation have altered floodplains from a mosaic of patches with variable IFI to a bimodal distribution. Relatively predictable flooding occurs in the connected forest, while inundation of the disconnected forest occurs only as the result of managed application of water. In semiarid floodplains, water is scarce except during the flood pulse. Ecosystem responses to the flood pulse are related to the IFI and other measures of flooding history that help describe spatial variation in ecosystem function.