Fatty acids with more than 22 carbon atoms (very-long-chain fatty acids; VLCFAs) are normal cellular components that have been implicated in the pathophysiology of a number of peroxisomal disorders. To date, however, essentially nothing is known regarding their biological activities. Ca2+ mobilization is an important intracellular signalling system for a variety of agonists and cell types. Given that several polyunsaturated long-chain fatty acids mobilize intracellular Ca2+ and that we have postulated that the VLCFAs may be involved in signal transduction, we examined whether the tetraenoic VLCFA induced Ca2+ mobilization in human neutrophils. We report that fatty acid-induced intracellular Ca2+ mobilization declined for fatty acid species of more than 20 carbon atoms, but increased again as the carbon chain length approached 30. This Ca2+ mobilization occurred independently of inositol 1,4,5-triphosphate production and protein kinase C translocation and involved both the release of Ca2+ from the intracellular stores and changes to the influx or efflux of the ion. We further observed that triacontatetraenoic acid [30:4(n-6)] mobilized Ca2+ from a thapsigargin-insensitive intracellular pool distinct from the thapsigargin-sensitive pools affected by arachidonic acid [20:4(n - 6)] or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). 20:4 (n - 6) induced strong superoxide production (chemiluminescence) which was inhibited by thapsigargin pretreatment. In contrast, fatty acid-induced superoxide production progressively declined as the carbon chain length increased beyond 20-22 carbon atoms. Further studies suggested that the thapsigargin-insensitive Ca2+ mobilization elicited by 30:4 (n - 6) was not related to oxyradical formation, while the thapsigargin-sensitive Ca2+ mobilization induced by 20:4 (n - 6) may be involved in the initiation but not necessarily the maintenance of superoxide production. In conclusion, this is the first report to demonstrate a biological activity for the VLCFA and indicates that 30:4 (n - 6) influences second messenger systems in intact cells that differ from those affected by long-chain fatty acids such as 20:4 (n - 6).