The perilipins are a family of polyphosphorylated proteins found exclusively surrounding neutral lipid storage droplets in adipocytes and steroidogenic cells. In steroidogenic cells, the cholesterol ester-rich lipid storage droplets are encoated with perilipins A and C. This study describes the dependence of perilipin levels on neutral lipid storage in cultured Y-1 adrenal cortical cells. The addition of fatty acids and cholesterol to the culture medium of Y-1 adrenal cortical cells greatly increased the storage of cholesterol esters and triacylglycerols concomitant with the formation of many new lipid storage droplets. The addition of fatty acids to the culture medium also produced a transient 6-fold increase in levels of perilipin A, but not C, mRNA, while much larger and stable increases in both perilipin A and C proteins were observed. The increases in perilipin protein levels were dependent upon the metabolism of fatty acids to triacylglycerol or cholesterol esters, since the incubation of cells with bromopalmitate, a poorly metabolized fatty acid, failed to yield large increases in lipid content or perilipin levels. Constitutive expression of epitope-tagged perilipins in transfected Y-1 adrenal cortical cells was regulated by lipid similarly to expression of the endogenous perilipins despite an absence of untranslated perilipin mRNA sequences in the expression constructs. Epitope-tagged perilipin A mRNAs were efficiently loaded with polyribosomes whether or not fatty acids were added to the culture medium; therefore, the increase in perilipin levels in the presence of fatty acids is likely due to factors other than increased translational efficiency. We suggest that the large increase in cellular perilipin levels upon lipid loading of cells is the result of post-translational stabilization of newly synthesized perilipins by stored neutral lipids.