To investigate the role of the monounsaturated n-9 fatty acids (MUFA) in the lipolytic activity of adipocytes, a study was carried out in which an increase in MUFA was produced in the tissues by two different methods; by the dietary enrichment of oleic acid or by producing an essential fatty acid deficiency syndrome. For this, forty-five male Sprague–Dawley rats were fed with a normal-energy diet and were subdivided into three groups. The diets varied in the type of dietary fat; palmitic acid, olive oil, or soyabean oil+palmitic acid. At the end of the study measurements were taken of weight, plasma leptin, tissue concentration of fatty acids, fat-cell size in the epididymal and the omental adipose tissues, adipocyte lipolytic activity of both tissues after stimulation with adrenaline, and the capacity of insulin to inhibit lipolysis. The baseline and adrenaline-stimulated lipolytic activity were greater and the anti-lipolytic capacity of insulin lower in the animals undergoing an increase in MUFA in the tissues (palmitic-acid and olive-oil diets). The area under the curve of glycerol, used as an indicator of lipolytic activity, was positively correlated with the concentration of MUFA and negatively with polyunsaturated fatty acids in the adipose tissues. It is concluded that an increase in tissue MUFA, however obtained, induces an increase in lipolytic activity.