The phenolic component of freeze-dried olive fruit was fractionated by high-performance liquid chromatography using ultraviolet, atmospheric pressure chemical ionisation (APCI) and electrospray ionisation (ESI) detection. The fractions together with several standards were tested for antioxidant activity in an aqueous and a lipid system. The negative ion mode of APCI and ESI showed less fragmentation than positive ion mode. The latter was generally more useful in obtaining fragmentation data and hence structural information. Some olive phenolics notably tyrosol exhibited a low ionisation efficiency in both APCI and ESI. There was no simple relationship between antioxidant activity and chemical structure. The ranking of antioxidant activity was strongly dependent on both the test system and on the substrate demonstrating the need to examine activity in both aqueous and lipid systems. Significant antioxidant activity was seen in most olive fractions and this was related to phenolic content. The kinetics of the oxidation process are complex and suggest that multiple pathways may be involved at different antioxidant concentrations.