1‐O‐Alkylglycerols (alkyl‐Gro), naturally occurring compounds abundant in shark liver oil, protect patients from radiotherapy side‐effects. However, the protection mechanism is not well understood. It might be mediated by alkyl‐Gro incorporation into pools of platelet‐activating factor (PAF) precursor and subsequent modification of PAF biosynthesis. Using a ³H‐labelled or unlabelled natural alkyl‐Gro mixture, in which prominent alkyl chains were C18:1(9) (54–65%), C16:1(7) (5–15.5%), and C16:0 (5–10%), we investigated the incorporation of alkyl‐Gro into phospholipids of human leukemic monocyte‐like THP‐1 cells. Incubation of cells for 24 h with [³H]alkyl‐Gro (10 μM) resulted in their incorporation into 1‐O‐alkyl‐2‐acyl‐sn‐glycero‐3‐phosphocholine (1097 ± 25.1 pmol/2×10⁶ cells) and into 1‐alkyl‐2‐acyl‐sn‐glycero‐3‐phosphoethanolamine (640.4 + 12.5 pmol/2×10⁶ cells) with a total yield of 6.5%. Such incorporation induced production of 1‐O‐[³H]alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine ([³H]PAF), which was increased after stimulation by the calcium ionophore A23187. HPLC analysis of the [³H]PAF molecular species indicated that the three major [³H]alkyl‐Gro were used for [³H]PAF synthesis in ratios similar to that of the mixture. Total production of biologically active PAF, as measured by the platelet‐aggregation bioassay, was also increased by alkyl‐Gro incorporation in resting (+20%) and in A23187‐stimulated (+59%) THP‐1 cells. HPLC analysis of the [³H]PAF produced in the presence of [³H]acetate, confirmed that levels of PAF, but not of its 1‐acyl analog, were increased by alkyl‐Gro incorporation in resting and stimulated cells. However, the rise in [³H]acetyl‐PAF, which resulted mainly from C16:0 PAF, was reduced by about 50% in the presence of the PAF‐receptor antagonist SR 27417, providing evidence that stimulation of total PAF synthesis was caused by the increase in the precursor pool and autocrine amplification of PAF‐induced PAF production. Thus, the supplementation of THP‐1 cells in culture with naturally occurring alkyl‐Gro led to the incorporation of alkyl‐Gro into ether‐containing phospholipids, which were subsequently used for PAF synthesis. Furthermore, alkyl‐Gro incorporation resulted in a significant rise in PAF production by THP‐1 cells under resting and stimulated conditions. These results may be of importance for modulating PAF production in several pathophysiological conditions, such as peroxysome deficiencies, that are associated with a lack of ether lipid synthesis.