The effects of the atmospheric oxygen concentration on the physical and chemical ignition behavior of biodiesel and diesel fuel blends were investigated using an optically accessible constant-volume combustion chamber (600 °C and 20 bar) equipped with a light-duty diesel injector. High-speed imaging was used to measure the transient cone angle and penetration length of the liquid spray jet as it developed within the chamber. Structural fluctuations of the developing spray jets were suggested to be a function of injector pin movement, with their frequency and amplitude being functions of the fuel viscosity and density. Physical and chemical ignition delay was analyzed through detection of excited formaldehyde and hydroxide chemiluminescence using a system of optical filters and photomultiplier tubes. Although biodiesel produced slightly longer physical ignition delays as a result of an extended atomization and evaporation process, its chemical ignition delay was much shorter than that of diesel, which is attributed to the long-chain hydrocarbon fuel structure and fuel oxygen availability. From an engine control and performance standpoint, the short ignition delay and robust heat release profile of biodiesel produced under simulated low-temperature combustion operating conditions demonstrates the compatibility of biodiesel for use in nextgeneration compression ignition engines.