Cytosolic phospholipase A₂ (cPLA₂) is a Ca²⁺-sensitive enzyme that has been implicated in insulin secretion in response to agents that elevate β-cell intracellular Ca²⁺ ([Ca²⁺]_i). We generated clones of the MIN6 β-cell line that stably underexpress cPLA₂ by transfection with a vector in which cPLA₂ cDNA had been inserted in the antisense orientation. Reduced expression of cPLA₂ was confirmed by Western blotting. The insulin content of cPLA₂-deficient MIN6 cells was reduced by ∼90%, but they showed no decrease in preproinsulin mRNA expression. Measurements of stimulus-dependent changes in [Ca²⁺]_i indicated that reduced expression of cPLA₂ did not affect the capacity of MIN6 cells to show elevations in Ca²⁺ in response to depolarizing stimuli. Perifusion experiments indicated that cPLA₂ underexpressing MIN6 pseudoislets responded to glucose, tolbutamide, and KCl with insulin secretory profiles similar to those of cPLA₂ expressing pseudoislets, but that secretion was not maintained with continued stimulus. Analysis of the ultrastructure of cPLA₂-deficient MIN6 cells by electron microscopy revealed that they contained very few mature insulin secretory granules, but there was an abundance of non–electron-dense vesicles. These data are consistent with a role for cPLA₂ in the maintenance of insulin stores, but they suggest that it is not required for the initiation of insulin secretion from β-cells.