Beta3 adrenoceptors (b3-AR) mediate a NO-dependent negative inotropic effect and are upregulated in the failing heart. As the role of these receptors in other cardiac diseases is unknown so far, we analyzed expressional and functional modulations of the b3 adrenergic pathway in human septic myocardium and a murine model of sepsis, a condition associated with myocardial depression. The causal role of the b3AR was tested in genetically-deleted mice and cardiomyocytes. b3AR and eNOS protein abundance was assessed by Western blotting in ventricles from mice at 0, 2, 4, 6, 8, 10, 12 and 24 h after injection with lipopolysaccharide (LPS, 10 mg/Kg, i.p. ; n = 25) or saline vehicle (n = 16), and in neonatal mouse ventricular myocytes (NMVM) incubated with conditioned medium from LPS-stimulated cultured macrophages (Mc-LPS+ medium). A similar analysis was performed in the myocardium of 8 patients who died from sepsis. Finally, the effects of BRL 37344, a (b3-preferential agonist, with or without L-NAME (NOS inhibitor) was analyzed by videomicroscopy on the contractility of NMVM incubated or not with Mc-LPS+ medium. In hearts of LPS-treated mice and in Mc-LPS+ NMVM, the b3-AR protein gradually increased, peaking at 340 ± 97 % of controls (p < 0.01) at 8 h and 320 ± 174 % of controls (p < 0.05) at 6 h, respectively. The levels of b3-AR were also increased (224 ± 11.2% of controls; p = 0.001) in hearts from septic patients (n = 8), compared to normal hearts (n = 15). eNOS was unchanged over time in mouse ventricular cells, and was increased to 295 ± 60.9% (p = 0.01) in the myocardium of septic patients. Stimulation of untreated NMVM with BRL37344 decreased the amplitude of contractile shortening to 19.1 ± 5.65% of baseline at 15 min (n = 23; p < 0.05). This response was unchanged by nadolol, a b1- b2-blocker, but abolished by bupranolol, a b1- b2- b3-blocker, or L-NAME (n = 9). Incubation in Mc-LPS+ medium potentiated the depressing effect of BRL37344 to -45.3 ± 4.78 % (n = 7; p < 0.05), that was partially reversed by L-NAME, to -23.71 ± 10.83%(n = 3). Accordingly, the cardiodepressant effect of BRL37344 was potentiated by Mc-LPS+ in wild-type (FVB) but not in b3-AR KO cardiomyocytes (P < 0.001 by ANOVA; n = 5-16). However, there was no difference in mortality after LPS injection in vivo between b3-AR KO and FVB. We conclude that b3-AR are upregulated in the murine and human myocardium during sepsis, and mediate an increased negative inotropic response to the b3 agonist, BRL37344. Activation of the b3-AR pathway by catecholamines may contribute to the myocardial dysfunction in sepsis.