The objective of this research project was to compare the emissions of oxides of nitrogen (NOx) from transit buses on as many as five different fuels and three standard transit duty cycles to establish if there is a real-world biodiesel NOx increase for transit bus duty cycles and engine calibrations. Prior studies have shown that B20 can cause a small but significant increase in NOx emissions for some engines and duty cycles. Six buses spanning engine build years 1998 to 2011 were tested on the National Renewable Energy Laboratory's Renewable Fuels and Lubricants research laboratory's heavy-duty chassis dynamometer with certification diesel, certification B20 blend, low aromatic [California Air Resources Board (CARB)] diesel, low aromatic B20 blend, and B100 fuels over the Manhattan, Orange County and UDDS test cycles. The buses selected represented the majority of the current national transit fleet as well as including hybrid and selective catalyst reduction (SCR) systems that are increasing penetration in the fleet.The engine emissions certification level had the dominant effect on NOx, with the kinetic intensity of the tested duty cycle being the secondary driving factor. The biodiesel effect on NOx emissions was not statistically significant for most buses and duty cycles for blends with certification diesel, with the exception being a 2008 model year bus. CARB fuel had many more instances of a statistically significant effect of biodiesel increasing NOx. SCR systems proved effective at reducing NOx to near the detection limit on all duty cycles and fuels, including B100. A hybrid system proved to significantly increase NOx emissions over a same model year bus with a conventional drivetrain and the same engine. As all but one test bus were equipped with diesel particulate filter aftertreatment PM emissions were negligible and trends could not be drawn. On the oldest sample bus without aftertreatment PM emissions were reduced with B20 blends. Fuel economy was not significantly changed by engine certification level except that the 2008 conventional bus had the best performance on all cycles while all other buses had very similar results on each cycle. All buses had lower fuel economy with increased kinetic intensity of the cycle.