Exogenous FA cause lipid accumulation in pre-adipocytes. We investigated whether the fat cells thus formed are metabolically distinct from adipocytes differentiated with standard methylisobutylxanthine, dexamethasone, and insulin (MDI) hormonal cocktail by comparing their expression of adipogenic genes, accumulation of TAG, lipogenesis, lipolysis, glucose uptake, and the effects of insulin on selected metabolic activities. Cells exposed to oleate began to accumulate TAG in parallel or prior to the induction of adipogenic genes, whereas cells treated with MDI expressed adipogenic genes before TAG accumulation. Oleate-treated fat cells also showed exaggerated basal lipolysis and weak response to insulin in both lipolysis regulation and glucose uptake. These findings were associated with increased basal phosphorylation of perilipin, increased Glut-1 but decreased Glut-4 expression, and reduced insulin-induced Akt phosphorylation. We suggest that this unique fat cell phenotype might be a mimetic of what can happen to fat cells formed in vivo under the influence of circulating FA and might be a useful model for in vitro studies of obesity-related insulin resistance in adipocytes.