We investigated the expression and activation of three MAPK subfamilies in the isolated perfused amphibian heart. ERK was detected as a 43 kDa band; p38‐MAPK was detected as a band corresponding to 38 kDa and JNKs were detected as two bands corresponding to 46 and 52 kDa, respectively. PMA induced the activation of the ERK pathway as assessed by determining the phosphorylation state of ERK and the upstream component MEK1/2. PD98059 abolished this activation. p38‐MAPK was phosphorylated by sorbitol (almost 12‐fold, maximal within 10–15 min) and JNKs were phosphorylated and activated by sorbitol or anoxia/reoxygenation (approximately 4‐ and 2.5‐fold, respectively). SB203580 completely blocked the activation of p38‐MAPK by sorbitol. These results indicate that the MAPK pathways activated by phorbol esters, hyperosmotic stress or anoxia/reoxygenation in the amphibian heart may have an important role in this experimental system.