Hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) regulate adipocyte lipolysis in rodents. Objective: To compare the roles of these lipases for lipolysis in human adipocytes. Design: Subcutaneous adipose tissue was investigated. HSL and ATGL protein expression were related to lipolysis in isolated mature fat cells. ATGL or HSL were knocked down by RNA interference or selectively inhibited and effects on lipolysis studied in differentiated preadipocytes or adipocytes derived from human mesenchymal stem cells (hMSC). Setting and subjects: Outpatient investigation. Subjects were all women, 12 lean controls, 8 lean with polycystic ovary syndrome and 27 otherwise healthy obese. Results: Noradrenaline-induced lipolysis was positively correlated with HSL protein levels (P<0.0001) but not with ATGL protein. Women with PCOS or obesity had significantly decreased noradrenaline-induced lipolysis and HSL protein expression but no change in ATGL protein expression. HSL knock-down by RNAi reduced basal and catecholamine-induced lipolysis. Knock-down of ATGL decreased basal lipolysis but did not change catecholamine-stimulated lipolysis. Treatment of hMSC with a selective HSL inhibitor during and/or after differentiation into adipocytes reduced basal lipolysis by 50% while stimulated lipolysis was inhibited completely. Conclusions: In contrast to findings in rodents, ATGL is of less importance than HSL in regulating catecholamine-induced lipolysis and cannot replace HSL when this enzyme is continuously inhibited. However both lipases regulate basal lipolysis in human adipocytes. ATGL expression, unlike HSL, is not influenced by obesity or PCOS.