We have studied the effect of three different fuels (fossil diesel fuel (EN590); rapeseed methyl ester (RME); and synthetic gas-to-liquid (GTL)) on heavy-duty diesel engine emissions. Our main focus was on nanoparticle emissions of the engine. Our results show that the particle emissions from a modern diesel engine run with EN590, GTL, or RME consisted of two partly nonvolatile modes that were clearly separated in particle size. The concentration and geometric mean diameter of nonvolatile nucleation mode cores measured with RME were substantially greater than with the other fuels. The soot particle concentration and soot particle size were lowest with RME. With EN590 and GTL, a similar engine load dependence of the nonvolatile nucleation mode particle size and concentration imply a similar formation mechanism of the particles. For RME, the nonvolatile core particle size was larger and the concentration dependence on engine load was clearly different from that of EN590 and GTL. This indicates that the formation mechanism of the core particles is different for RME. This can be explained by differences in the fuel characteristics.