The contributions of phospholipid headgroup structure, diacylglycerol, and Ca2+ in regulating the interaction of protein kinase C beta II with membranes or detergent/lipid mixed micelles were examined. Binding measurements revealed that, in the absence of diacylglycerol, protein kinase C displays no significant selectivity for headgroup structure other than change: the enzyme binds with equal affinity to phosphatidyl-L-serine, phosphatidyl-D-serine, and other monoanionic lipids such as phosphatidylglycerol. In contrast, selectivity for headgroup occurs in the presence of diacylglycerol. This second messenger increases the affinity of protein kinase C for phosphatidyl-L-serine-containing membranes or micelles by 2 orders of magnitude, but has only moderate effects on the affinity of protein kinase C for surfaces containing other anionic lipids. Ca2+ does not affect the diacylglycerol-mediated increase in protein kinase C's affinity for phosphatidylserine, but does increase the enzyme's affinity for acidic phospholipids. Lastly, ionic strength studies reveal that electrostatic interactions are the primary driving force in the interaction of protein kinase C with membranes. In the absence of either diacylglycerol or phosphatidylserine, these interactions are sufficiently weak that little binding occurs at physiological ionic strength; thus, protein kinase C is unlikely to translocate to the plasma membranes in the absence of diacylglycerol, even if intracellular Ca2+ levels are high. Our data reveal that, although there is no specificity for binding acidic lipids in the absence of diacylglycerol, specific structural elements of the L-serine headgroup are required for the high-affinity binding of protein kinase C to diacylglycerol-containing membranes.