Adipocytes from post-menopausal females have higher basal lipolytic rates than pre-menopausal females, which contributes to increased risk of developing dyslipidemia following menopause. The purpose of this study was to delineate cellular mechanisms affecting adipose tissue function in the ovariectomized (OVX) mouse and also determine if physical activity or estrogen supplementation alter any detected changes. Female C57/Bl6 mice were placed into SHAM, OVX sedentary (OVX), OVX exercise (OVX-Ex), and OVX sedentary + 17β-estradiol (OVX + E2) groups. Visceral fat mass, glycerol, and NEFA levels were significantly higher in OVX mice compared to SHAM animals, but were not elevated in the E2-treated animals. Voluntary running failed to change circulating levels of glycerol or NEFA in OVX mice, but did partially attenuate the increase in visceral fat mass. Adipose triglyceride lipase (ATGL) protein content was significantly elevated in visceral fat from OVX and OVX-Ex groups compared to SHAM, while ATGL–CGI-58 interaction was significantly higher in OVX than SHAM and OVX + E2 mice. No significant differences in HSL phosphorylation were detected between groups, however, ERK1/2 phosphorylation was significantly elevated in the OVX mice. To determine if ERK1/2 function was critical for the increased glycerol levels, visceral fat was treated with MEK inhibitor PD98059, with no differences in glycerol release detected. Perilipin protein content was decreased significantly in OVX and OVX-Ex mice compared to SHAM. Thus, these data suggest that increased ATGL signaling and reduced perilipin protein content may contribute to increased NEFA and glycerol levels in OVX mice, which are attenuated with E2 treatment, but not by exercise. J. Cell. Biochem. 110: 420–427, 2010. © 2010 Wiley-Liss, Inc.