Estradiol benzoate (E2) increases plasma lipids in hypothalamic obese, functionally castrated (OFC), obese laying (OL), and control laying hens (CONT). However, E2 reduces fattiness in OFC but not in OL or CONT hens. Antiestrogen, such as tamoxifen (TAM), reduces plasma lipids in OL and CONT, but not in OFC, hens and has no effect on fattiness in any of them. Apolipoprotein VLDL-II (apo-VLDL-II), lipoprotein lipase (LPL), and rate of lipolysis may mediate these estrogenic effects. In the present study, effects of E2 and TAM on fattiness, plasma apo-VLDL-II, in vitro lipolysis, and LPL activity in postheparin plasma and abdominal adipose tissue (AAT) were determined in OFC, OL, and CONT hens. Basomedial hypothalamic lesions were performed in 3-month-old White Leghorn hens. At the static phase, 10 months later, OFC OL, and CONT hens were divided into three subgroups and injected IM on alternate days, with either 2 mg E2/kg b.wt., 10 mg TAM/kg, or vehicle corn oil, for 5 weeks. In OL and OFC hens, body and AAT weights were higher than in CONT poullets. Food intake and ovarian weight were similar in OL and CONT, higher than in OFC hens. Plasma LPL activity was higher, whereas plasma apo-VLDL-II and stimulated lipolysis were lower in OFC than in OL and CONT hens. In OFC hens LPL activity per unit of AAT was half than in OL and CONT. Total LPL activity in AAT was similar in OFC and CONT and higher in OL hens. Levels of basal lipolysis were similar in all experimental hens. TAM did not affect any of the measured parameters in OFC hens. In OL and CONT hens, TAM depressed apo-VLDL-II, increased plasma LPL activity, but had no effects on AAT LPL activity, on stimulated lipolysis, or fattiness. E2 increased apo-VLDL-II to similar levels in all groups and reduced LPL activity in plasma and AAT of obese hens. Only in OFC hens did E2 enhance basal and stimulate lipolysis and reduce FI and fattiness. We conclude that in adult laying hens, unlike in cockerels and juvenile hens, estrogen reduces lipid incorporation in fat depots by enhancing apo-VLDL-II production that reduces plasma and AAT LPL activity. This may increase lipoprotein available for incorporation into developing yolks. The lack of estrogen in OFC hens reduces circulating apo-VLDL-II and thus increases LPL activity and amount of fat depots.