1. Gelatinases (matrix metalloproteinase (MMP)-2 and MMP-9) might play a role in the development and structural organization of adipose tissue. Obese mice were subjected to caloric restriction (from 82 to 27 kJ/day) for 6 weeks, in order to investigate the effect of drastic weight loss on gelatinase mRNA, protein and activity levels. 2. Caloric restriction resulted in significantly lower bodyweight, as well as subcutaneous (SC) and gonadal (GON) fat mass (all P < 0.001). 3. The expression of MMP-2 mRNA was significantly upregulated in both SC (3.3-fold) and GON (2.2-fold) adipose tissues; however, without significant effect on total MMP-2 protein levels in fat tissues or in plasma. In contrast, MMP-9 mRNA expression in SC or GON fat was not affected by caloric restriction, whereas protein levels were reduced in SC and GON fat, but not in plasma. Zymography showed significantly elevated levels of active MMP-2 in both SC and GON adipose tissues, whereas active MMP-9 levels were reduced in GON fat. 4. These findings imply that the evaluation of the role of gelatinases in obesity and metabolic disorders requires the determination of both antigen and activity levels in plasma and fat tissues.