Previous studies with phospholipid monolayers revealed a large decrease in the activity of phosphoinositide-specific phospholipase C-delta(1) (PLC-delta(1)) which catalyses the hydrolysis of PtdIns(4, 5)P(2) as lateral pressure is applied to the membrane. If stress on the membrane is the sole inhibitor of PLC-delta(1) activity, the enzyme must penetrate the membrane surface to engage its substrate. To test the effect on PLC-delta(1) activity of lipid packing in the absence of a directional stress, we examined the effects of increasing hydrostatic pressure on enzymic activity. We find that, in contrast with monolayer studies, increasing lipid packing by hydrostatic pressure does not affect membrane binding and increases enzymic activity by 90% in going from atmospheric pressure to 10(8) Pa (approx. 1000 atm). The increase in activity could be accounted for mainly by electrostriction of water around the multiply-charged product. Our results show that when there is no net stress on the monolayer, lipid packing does not alter PLC-delta(1) activity, possibly because penetration of the enzyme into the membrane surface is shallow. We suggest that, in biological membranes, the activity of this and possibly other interfacial proteins is independent of headgroup packing.