Plasminogen activator inhibitor-I (PAI-I) is the main physiological inhibitor of tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator. Recent studies in murine models have yielded apparently conflicting data on a potential role of PAI-I in adipose tissue development and obesity. To reinvestigate this issue, we have rederived PAI-I deficient (PAI-I-/-) and wild-type (WT) mice and generated true littermates in a 81.25% C57Bl/6: 18.75% 129 SV genetic background. Male 5-week-old PAI-I-/- and WT mice were kept on a high fat diet (20.1 kJ/g) for 15 weeks. Body weight gain was comparable for both genotypes, and at the time of sacrifice total body weights (39 &PLUSMN; 1.1 versus 41 &PLUSMN; 1.2 g) as well as the weights of subcutaneous (SC, 1,520 &PLUSMN; 10 versus 1,480 &PLUSMN; 10 mg) adipose tissue were not significantly different. In contrast, the gonadal (GON, 1,900 &PLUSMN; 43 versus 1,510 &PLUSMN; 86 mg, p < 0.005) tissue mass was larger in PAI-I-/- mice. Plasma levels of insulin, leptin, glucose, triglycericles, total, HDL and LDL cholesterol were comparable for both genotypes. Immunohistochemical analysis of SC and GON adipose tissues did not reveal differences in adipocyte size or number between both genotypes, whereas blood vessel density was also comparable for GON fat but lower in SC fat of WT mice. Thus, this study in littermate mice on high fat diet did not reveal an effect of PAI-I deficiency on body weight, and a differential effect on SC and GON adipose tissue.