The potential of biodiesel as a viable alternative to petroleum diesel has been driving experimental efforts to insure efficient, high-power, and low emissions operation for many years. Literature is rich with discussion about the differences in operation between biodiesel and petroleum diesel; often, however, these discussions focus on time averaged results that may not detect subtle differences in cycle-to-cycle operation. This aspect has motivated this research study, which compares certain combustion aspects of both fuels on a cycle-by-cycle basis. Thus, the objective of this experimental study is to link fuel property differences between biodiesel and petroleum diesel fuels to potential differences in cycle-to-cycle variability. Steady-state operation of a medium-duty diesel engine at nine different operating conditions, for each fuel, is discussed. In-cylinder pressure measurements, and the resulting calculated apparent heat release rates are the primary tools used to interpret the differences and how the fuel properties ultimately affect the cycle-to-cycle combustion variability of the engine. In general, cycle-to-cycle variability with biodiesel is on the same order as that of petroleum diesel fuel, which for diesel engines is already very low. This study finds that the differences in certain fluid properties, and their resulting manifestations on mixture preparation prior to combustion, are the primary sources for the observed differences in cycle to cycle variability. Based on these differences, predictions are made as to which fuel would exhibit relatively higher cyclic combustion variability. A number of parameters indicative of combustion variation are calculated to quantify observed differences and the results show general agreement with the predictions.