Perilipin A is a key regulator of triacylglycerol storage and hydrolysis in adipocytes; phosphorylation of perilipin A by protein kinase A facilitates maximal lipolysis. Chronic stimulation of lipolysis in 3T3-L1 adipocytes causes large perinuclear lipid droplets to fragment into myriad dispersed perilipin A-covered microlipid droplets. In cultured fibroblasts stably expressing ectopic perilipin A, clustered lipid droplets disperse throughout the cytoplasm upon incubation of the cells with forskolin and isobutylmethylxanthine (IBMX) to elevate levels of cAMP and activate protein kinase A, mirroring events observed in adipocytes. Furthermore, diethylumbelliferyl phosphate inhibits stimulated lipolysis but not the dispersion of lipid droplets, suggesting that products of lipolysis are not required for this remodeling process. We hypothesized that protein kinase A-mediated phosphorylation of perilipin A triggers the remodeling of lipid droplets. The mutation of serine 492 of perilipin A to alanine prevented the dispersion of clustered lipid droplets in fibroblasts stably expressing the mutated perilipin upon incubation with forskolin and IBMX. In contrast, the substitution of serines 81, 222, 276, or 433 with alanine, either singly or in combinations, did not affect the protein kinase A-mediated remodeling of lipid droplets. Interestingly, substitution of serines 433, 492, and 517 of perilipin A with glutamic acid residues blocked the dispersion of clustered lipid droplets in cells incubated with forskolin and IBMX, indicating that the addition of a negative charge does not mimic a phosphate group. We conclude that protein kinase A-mediated phosphorylation of serine 492 of perilipin A drives the fragmentation and dispersion of lipid droplets.Adipose tissue contains the largest energy reserve in the body, stored as triacylglycerol in the intracellular lipid droplets of adipocytes. Triacylglycerol stores are hydrolyzed to mobilize energy during fasting and extended exercise. Catecholamines bind to ␤-adrenergic receptors on the plasma membranes of adipocytes to initiate a G-protein-mediated signaling cascade that activates adenylyl cyclase, thus increasing intracellular cAMP levels. Protein kinase A (or cAMP-dependent protein kinase) is then activated and phosphorylates several proteins required for the hydrolysis of triacylglycerol and consequent mobilization of fatty acids. Following phosphorylation by protein kinase A, hormone-sensitive lipase translocates from the cytosol onto lipid droplets (1-5), where it hydrolyzes triacylglycerol and diacylglycerol (6 -8). Perilipin A is another major substrate for protein kinase A (9, 10).Perilipin A localizes to lipid droplets in adipocytes and plays roles in facilitating both the storage and hydrolysis of triacylglycerol. In adipocytes, two isoforms of perilipin are derived from the alternate splicing of mRNA transcribed from a single gene (10, 11); perilipin A is the predominant protein isoform, whereas perilipin B is a minor isoform. Perilipins A and B share a common sequ...