The rapid increase in palm oil production over the last 20 years has made this oil the most important in the world. Palm oil produced in the mesocarp of the oil palm (Elaeis guineensis) fruit contains primarily palmitic (44%) and oleic (39%) acids with only a small proportion of polyunsaturated fatty acids. As a prelude to detailed biochemical analysis of the fatty acid biosynthesis of oil palm, we examined β-ketoacyl-ACP synthase (KAS) II activity, and evidence is presented on its importance in palmitic acid accumulation. In this study, three different oil palm breeding materials were screened for KAS II and fatty acid compositions. Collectively, the results strongly suggest that KAS II plays an important role in determining the relative amounts of C16 and C18 fatty acids in oil palm. KAS II activity was profiled through various stages of fruit development from 12 to 22 weeks after anthesis (WAA). The data depicted a pattern of progressive increase in KAS II activity similar to that of triacylglycerol synthesis in the oil palm mesocarp which started at 15 WAA and reached a maximum around 20 WAA. The enzyme was purified to homogeneity (>9000-fold purification) using a combination of ion exchange on CM-Sepharose, HR-DEAE, hyroxyapatite and affinity chromatography (ACP-Sepharose). Activity of the purified enzyme was inhibited by the chelating agent ethylene diamine tetra-acetate (EDTA) (1 M), but the inhibitory action could be overcome by divalent cations such as Mn²⁺ or Mg²⁺ (10 mM). Optimal activity was observed at pH 4.5. The data represent the first contribution to the biochemical characterisation of oil palm KAS II activity associated with fatty acid biosynthesis.