Bioethanol, produced from renewable sources, is promoted as a fuel in higher concentrations in newer flexible fuel engines, and in lower concentrations in the general fleet. Introduction of a blend of 85% ethanol with gasoline (E85) at a competitive price in the Czech Republic has, however, spontaneously resulted in this fuel being used in “ordinary” engines not adapted for this fuel. This study investigates the operation of a typical gasoline car with fuel injection and three-way catalyst on gasoline, E85, and additionally on a blend of 85% n-butanol with gasoline, as butanol features better material compatibility than ethanol. The car was equipped with a portable, on-board emissions monitoring system and driven along a route comprising city and rural roads, including hills. Multiple runs were made on each fuel to verify test-to-test repeatability. The engine control unit has remarkably well adapted itself, over tens of km of driving, to both E85 and n-butanol by increasing the fuel injector pulse width, so that the engine was operated mostly at stoichiometric ratio on all fuels. Exhaust temperatures at tailpipe were comparable for all fuels. Operation on E85 and n-butanol blend has resulted, compared to gasoline, in no adverse effects on HC, lower emissions of CO and higher emissions of NOx, likely resulting from changes in air-fuel ratio. E85 had no effects on particle mass and reduced particle length (indicator of nanoparticles), while n-butanol has increased particle mass and had no adverse effects on particle length.