A simple and sensitive method for direct and continuous monitoring of free fatty acid (FFA) release, by measuring the pH-sensitive change in relative fluorescence intensity of seminaphthofluorescein (SNAFL-1) is described. The method was designed to use a small number of adipocytes isolated from fat pads of rats and biopsy specimens of horses for the detection of decreasing pH in fat cell suspensions caused by released FFA into the incubation medium. Species specific differences of lipolysis were demonstrated when adipocytes of rats and horses are incubated with stimulators or inhibitors of lipolysis. Norepinephrine (NE) stimulated lipolysis in fat cells of rats whereas adipocytes of horses showed a measurable release of FFA when concomitantly incubated with NE and adenosine deaminase (ADA) or NE and 8-Phenyltheophylline (8-PT), respectively). The incubation of equine fat cells with NE and ADA did not influence the antilipolytic response to insulin. The method described enables micro-scaled in vitro studies on lipolytic activity.