The role of protein kinase C (PKC) in the regulation of store operated Ca²⁺ entry (SOCE) is rather controversial. Here we used Ca²⁺ imaging, biochemical, pharmacological and molecular techniques to test if Ca²⁺-independent phospholipase A₂ beta (iPLA₂), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMC). We found that inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMC. Activation of PKC by the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca²⁺ stores and triggered Ca²⁺ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA₂ activity and Ca²⁺ entry that were absent in SMC in which iPLA₂ was inhibited by specific chiral enantiomer of bromoenol lactone, S-BEL. Moreover, we found that PKC regulates TG- and OAG-induced Ca²⁺ entry only in proliferating SMC, which correlates with the expression of specific PKC isoform. Molecular down regulation of PKC impaired TG- and OAG-induced Ca²⁺ influx in proliferating, but had no effect in confluent SMC. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve depletion of Ca²⁺ stores, as well as direct PKC-dependent activation of iPLA₂ resulting in a complex regulation of SOCE in proliferating and confluent SMC.