The number of heavy-duty vehicles using alternative fuels such as compressed natural gas (CNG) and new low-sulfur diesel fuel formulations and equipped with after-treatment devices are projected to increase. However, few peer-reviewed studies have characterized the emissions of particulate matter (PM) and other toxic compounds from these vehicles. In this study, chemical and biological analyses were used to characterize the identifiable toxic air pollutants emitted from both CNG and low-sulfur-diesel-fueled heavy-duty transit buses tested on a chassis dynamometer over three transient driving cycles and a steady-state cruise condition. The CNG bus had no after-treatment, and the diesel bus was tested first equipped with an oxidation catalyst (OC) and then with a catalyzed diesel particulate filter (DPF). Emissions were analyzed for PM, volatile organic compounds (VOCs; determined on-site), polycyclic aromatic hydrocarbons (PAHs), and mutagenic activity. The 2000 model year CNG-fueled vehicle had the highest emissions of 1,3-butadiene, benzene, and carbonyls (e.g., formaldehyde) of the three vehicle configurations tested in this study. The 1998 model year diesel bus equipped with an OC and fueled with low-sulfur diesel had the highest emission rates of PM and PAHs. The highest specific mutagenic activities (revertants/microg PM, or potency) and the highest mutagen emission rates (revertants/mi) were from the CNG bus in strain TA98 tested over the New York Bus (NYB) driving cycle. The 1998 model year diesel bus with DPF had the lowest VOCs, PAH, and mutagenic activity emission. In general, the NYB driving cycle had the highest emission rates (g/mi), and the Urban Dynamometer Driving Schedule (UDDS) had the lowest emission rates for all toxics tested over the three transient test cycles investigated. Also, transient emissions were, in general, higher than steady-state emissions. The emissions of toxic compounds from an in-use CNG transit bus (without an oxidation catalyst) and from a vehicle fueled with low-sulfur diesel fuel (equipped with DPF) were lower than from the low-sulfur diesel fueled vehicle equipped with OC. All vehicle configurations had generally lower emissions of toxics than an uncontrolled diesel engine. Tunnel backgrounds (measurements without the vehicle running) were measured throughout this study and were helpful in determining the incremental increase in pollutant emissions. Also, the on-site determination of VOCs, especially 1,3-butadiene, helped minimize measurement losses due to sample degradation after collection.