Future automotive fuels are expected to contain significant quantities of bio-components. This poses a great challenge to the designers of novel low-CO2 internal combustion engines because biofuels have very different properties to those of most typical hydrocarbons. The current article presents results of firing a direct-injection spark-ignition optical research engine on ethanol and butanol and comparing those to data obtained with gasoline and iso-octane. A multihole injector, located centrally in the combustion chamber, was used with all fuels. Methane was also employed by injecting it into the inlet plenum to provide a benchmark case for well-mixed “homogeneous” charge preparation. The study covered stoichiometric and lean mixtures (λ = 1.0 and λ = 1.2), various spark advances (30–50° CA), a range of engine temperatures (20–90°C), and diverse injection strategies (single and “split” triple). In-cylinder gas sampling at the spark-plug location and at a location on the pent-roof wall was also carried out using a fast flame ionization detector to measure the equivalence ratio of the in-cylinder charge and identify the degree of stratification. Combustion imaging was performed through a full-bore optical piston to study the effect of injection strategy on late burning associated with fuel spray wall impingement. Combustion with single injection was fastest for ethanol throughout 20–90°C, but butanol and methane were just as fast at 90°C; iso-octane was the slowest and gasoline was between iso-octane and the alcohols. At 20°C, λ at the spark plug location was 0.96–1.09, with gasoline exhibiting the largest and iso-octane the lowest value. Ethanol showed the lowest degree of stratification and butanol the largest. At 90°C, stratification was lower for most fuels, with butanol showing the largest effect. The work output with triple injection was marginally higher for the alcohols and lower for iso-octane and gasoline (than with single injection), but combustion stability was worse for all fuels. Triple injection produced a lower degree of stratification, with leaner λ at the spark plug than single injection. Combustion imaging showed much less luminous late burning with tripe injection. In terms of combustion stability, the alcohols were more robust to changes in fueling (λ = 1.2) than the liquid hydrocarbons.