At concentrations above 10 ng ml⁻¹, the tumour promoter thapsigargin stimulates the release of radioactivity from [³H]‐arachidonic acid‐labelled macrophages harvested from rat peritoneal cavity. The release of radioactivity from prelabelled macrophages was augmented more than additively when the cells were incubated in the medium containing both thapsigargin (10 ng ml⁻¹) and other tumour promoters (10 ng ml⁻¹), such as 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), teleocidin and aplysiatoxin. Thapsigargin required extracellular Ca²⁺ for the stimulation of arachidonic acid release, while TPA did not. Cytoplasmic free calcium level was increased by thapsigargin treatment but not by TPA treatment. An inhibitor of protein kinases, H‐7 inhibited the effect of TPA dose‐dependently, whereas H‐7 did not inhibit that of thapsigargin. These results suggest that thapsigargin stimulates arachidonic acid release by a mechanism different from that of TPA, viz by acting as a selective Ca²⁺ mobilizer, but not by activating protein kinase C as TPA does. 1988 British Pharmacological Society