The relationship between isoproterenol-induced lipolysis and the phosphorylation of perilipin and hormone-sensitive lipase (HSL) was examined using cell-free systems consisting of lipid droplets isolated from rat fat cells and HSL, and/or trioleoylglycerol emulsified with gum arabic and HSL. Isoproterenol was found to stimulate lipolysis in the cell-free system with the lipid droplets without an increase in the phosphorylation of either perilipin or HSL. On the other hand, no stimulation of lipolysis was found in the cell-free system containing lipid droplets despite increases in the phosphorylation of perilipin and HSL. In the cell-free system consisting of trioleoylglycerol emulsified with gum arabic and HSL, neither isoproterenol nor increases in the phosphorylation of perilipin and HSL accelerated lipolysis, These results suggest that isoproterenol-induced lipolysis may not be mediated through the phosphorylation of perilipin and HSL, and may rather be dependent on the substrate of HSL.