Clinical studies have documented gender differences in the pattern of LV hypertrophy, but the mechanisms are yet poorly defined. This study aimed to determine whether (i) severe pressure overload altered expression and/or activity of cardiac constitutive NOS (NOS1 and NOS3), (ii) these changes were modulated according to gender. Analyses were performed from 0.4 to 20 weeks (wk) after thoracic aortic constriction (TAC) in male (M) and female (F) Wistar rats. M-TAC exhibited early signs of cardiac dysfunction as shown by echocardiographic and LV-end diastolic pressure measurements conversely to F-TAC, which exhibited higher LV hypertrophy (+96% vs M-TAC at 20 wks, p<0.05). Following TAC, cardiac NOS1 expression was rapidly induced (0.4 wks) and stable afterwards in males (p<0.05 vs sh) whereas it was delayed in females. Accordingly specific NOS1 activity was increased by 2wks in M-TAC (+122%, p<0.001 vs sh) and only by 20wks in F-TAC (+ 220% p<0.001 vs sh). NOS1 activity was correlated with NOS1 level. Regarding cardiac NOS3, the expression was unaffected by TAC and the decrease in activity observed at early and late times in M-TAC and F-TAC, respectively, is shown to be related to the level of NOS3 allosteric regulator caveolin-1. The data demonstrated a unique gender-dependent regulation of the constitutive NOSs in response to TAC in rats, such a difference might play a role in the gender-dependent adaptability of the heart in response to pressure overload.