Myocardial stretch elicits a biphasic increase in developed force with a first rapid and second slow force response (SFR). The rapid phase is due to an increase in myofilament Ca²⁺ responsiveness while the SFR, analyzed herein, is ascribed to a progressive increase in Ca²⁺ transients. Experiments were performed in cat papillary muscles to further elucidate the signaling pathway underlying the SFR. While the SFR was diminished by BQ123, a similar endothelin (ET) -1-induced increase in force was not affected (23±2 % vs. 23±3%, NS). Instead, BQ123 suppressed the contractile effects of either ET-2 or ET-3 (21±2 and 25±3 vs. -1±1 and -7±3 % respectively; P< 0.05), suggesting that either of these isoforms, but not ET-1, was involved in the SFR. Each isoform activated the Na⁺/H⁺ exchanger 1) NHE-1 increasing Na⁺_i by 2.0±0.1, 2.3±0.1 and 2.1±0.4 mmol/L for ET-1, ET-2 and ET-3 respectively (P<0.05). The NHE-1 inhibitor HOE 642 prevented the increases in force and Na⁺_i induced by all the ETs, but only ET-2 and ET-3 effects were sensitive to BQ123. Real time RT-PCR measurements of prepro ET-1, ET-2 and ET-3 mRNA were performed previous to and at 5, 15 and 30 minutes after stretch. No changes in ET-1 or ET-2 but an increase of ~60 % in ET-3 mRNA after 15 minutes of stretch were detected. Stretched-induced ET-3 mRNA up-regulation and its mechanical counterpart were suppressed by AT₁ receptors blockade by losartan. Therefore, these data suggest a role for AT₁-mediated ET-3 released in the early activation of the NHE-1 that follows myocardial stretch.