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The aim of this study is to analyze the influence of horizontal geometric design and vehicle operation on fuel consumption and gas emissions produced by passenger cars. Continuous speed data were collected along 15 two-lane rural road sections. Different horizontal alignment indexes and operational variables were obtained. Fuel consumption and gas emissions were estimated by applying the VT-Micro model. The results showed that Curvature Change Rate and average speed have a significant impact on average fuel consumption and gas emissions. Finally, different regression models were calibrated based on these variables to estimate fuel consumption [l/100 km] and emissions [gr/km]. These results could be the basis to incorporate environmental sustainability principles into road design guidelines, since currently they do not take into account the environmental impact related to highway geometric design.