In white adipose tissue, lipolysis can occur by hormone-sensitive lipase (HSL)-dependent or HSL-independent pathways. To study HSL-independent lipolysis, we placed HSL-deficient mice in conditions of increased fatty acid flux: beta-adrenergic stimulation, fasting, and dietary fat loading. Intraperitoneal administration of the beta(3)-adrenergic agonist CL-316243 caused a greater increase in nonesterified fatty acid level in controls (0.33 +/- 0.05 mmol/l) than in HSL(-/-) mice (0.12 +/- 0.01 mmol/l, P < 0.01). Similarly, in isolated adipocytes, lipolytic response to CL-316243 was greatly reduced in HSL(-/-) mice compared with controls. Fasting for <or=48 h produced normal mobilization and oxidation of fatty acids in HSL(-/-) mice, as judged by similar values of respiratory quotient and oxygen consumption as in HSL(+/+) controls. In isolated adipocytes, lipolysis in the absence of beta-adrenergic stimulation was 1.9-fold greater in HSL(-/-) than in HSL(+/+) cells (P < 0.05), increasing to 6.5-fold after fasting (P < 0.01). After 6 wk of a fat-rich diet containing 31.5% of energy as lipid, weight gain of HSL(-/-) mice was 4.4-fold less than in HSL(+/+) mice (P < 0.01), and total abdominal fat mass was 5.2-fold lower in HSL(-/-) than in HSL(+/+) mice (P < 0.01). In white adipose tissue, HSL is essential for normal acute beta-adrenergic-stimulated lipolysis and permits normal triglyceride storage capacity in response to dietary fat loading. However, HSL-independent lipolysis can markedly increase during fasting, both in isolated adipocytes and in intact mice, and can mediate a normal flux of fatty acids during fasting.