The Midwest is one of the most important production areas for corn and soybean worldwide, but also comprises remnants of natural tallgrass prairie vegetation. Future predictions suggest that corn (Zea mays L.) and soybean (Glycine max (L.) Merr.) production in the Midwest may be limited by precipitation and temperature due to climate change. Cross-biome long-term studies in situ are needed to understand carbon assimilation and impact of climate change on the entire region. In this study, we investigated the differences of gross primary production (GPP) and net ecosystem production (NEP) among typical (agro-) ecosystems of corn, soybean and tallgrass prairie from eddy flux stations from 2006 to 2015 under contrasting weather conditions. Corn had the highest annual GPP and NEP with 1305 and 327 g Cm-2 yr(-1), while soybean had significantly lower GPP and NEP with 630 and 34 g Cm-2, excluding additional carbon loss by yield. Corn and soybean NEP was linear related (p < 0.05) to leaf area index (LAI), height or phenological stage, confirming the strong link between plant growth and ecosystem carbon balance. Tallgrass prairie had average values of GPP and NEP of 916 and 61 g Cm-2 yr(-1), excluding loss of carbon by annual burning. Thus, prairie GPP and NEP were significantly lower than corn, but significantly higher than soybean. Probably the long fallow period on cropland, which enhanced heterotrophic respiration, and the low carbon assimilation of soybean reduced its overall carbon balance. In total, the corn-soybean agroecosystem acted as a carbon source due to carbon loss by yield removal. Values for GPP and NEP were reflected in inherent water use efficiency (IWUE*) and light use efficiency (LUE) among the agroecosystems. In addition, IWUE*, LUE or GPP of crops and tallgrass prairie were linearly related (p < 0.05) to precipitation, volumetric soil water content (VWC) and maximum air temperature. Air temperature increased IWUE* in both, cropland and prairie vegetation. However, rainfall and VWC affected crops and prairie vegetation differently: while excessive rainfall and VWC reduced GPP or IWUE* in cropland, prairie vegetation GPP and LUE were adversely affected by reduced VWC or precipitation. Future measures of climate change adaption should consider the contrasting effects of precipitation and VWC among the different agro-ecosystems in the Midwestern USA. (C) 2016 Elsevier B.V. All rights reserved.