Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) regulate adipocyte lipolysis in rodents. The purpose of this study was to compare the roles of these lipases for lipolysis in human adipocytes. 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 (RNAi) or selectively inhibited, and effects on lipolysis were studied in differentiated preadipocytes or adipocytes derived from human mesenchymal stem cells (hMSC). Subjects were all women. There were 12 lean controls, 8 lean with polycystic ovary syndrome (PCOS), and 27 otherwise healthy obese subjects. We found that norepinephrine-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 norepinephrine-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 in adipocytes reduced basal lipolysis by 50%, but stimulated lipolysis was inhibited completely. 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.