Proteins of the annexin/lipocortin family have been claimed to mediate the anti-inflammatory action of glucocorticosteroids by the inhibition of phospholipases A2. This hypothesis has been challenged by the finding that annexins do not directly interact with the enzyme in a classical enzyme/inhibitor behavior, but more likely block the access of the phospholipase A2 to its substrate by binding to phospholipids. Because former studies with skin phospholipase A2 suggested a specific regulation by annexin-1, we investigated the substrate dependence of this effect. For this purpose phospholipase A2 activities in human epidermis and dermis homogenates were measured in the presence of various amounts of annexins-1, -2, or -5. The respective annexin was preincubated in separate series either with the substrate or with the enzyme. We found a partial inhibition of both epidermal and dermal phospholipase A2 activities with all annexins tested (annexin-5 >> annexin-2 > annexin-1). The inhibitory effect was absolutely dependent on the annexin/phospholipid ratio and occurred only at very high annexin concentrations relative to the amount of substrate. Our data demonstrate that the inhibition of human skin phospholipase A2 by annexins depends on the substrate concentrations, as has been shown for phospholipases A2 of other origins as well. All observations can be explained by the current "substrate depletion model" characterizing the indirect effects of annexins on phospholipase A2 activities. It is therefore rather unlikely that annexins are directly involved in the regulation of phospholipase A2 activity of human skin under physiologic conditions.