The validation of detailed models, in terms of SO3 formation in large marine engines operating on sulfur-containing heavy fuel oils (HFOs), relies on experimental work. The requisite is addressed in the present work, where SO3 is measured in the exhaust gas of an 80 kW medium-speed single-cylinder HFO-fuelled test engine. SO3 formation is triggered by running the engine at altered operational conditions and speeds within 1050−1500 rpm. The test engine does not represent a large low-speed marine engine; however, the nature of high-temperature SO3 formation may well be explored with the current engine and serve as reference for further modeling studies. SO3 is measured using a continuous SO3 monitor from PENTOL GmbH. The monitor offers online SO3 readings and short sampling times, in contrast to other extractive methods. The measurement is based on SO3 capture in isopropanol prior to chemical conversion and indirect detection via light absorption in a photometer. Present results show that SO3 formation is favored by elevated pressure histories, premixed combustion, and reduced speeds. The fraction of fuel sulfur converted to SO3 is measured to be on the order of 0.5%−2.4%, corresponding to 4−14 ppmv. SO3 and NOx are not comparable, according to thermodynamic considerations, yet both species involve the radical pool and are studied in parallel. Resulting emissions of SO3 and NOx in the exhaust gas follow a comparable trend throughout the experiments.