We previously reported that both hypoxia and hypoxia followed by reoxygenation (hypoxia/reoxygenation) rapidly activate Src family tyrosine kinases and p21ras in cultured rat cardiac myocytes. This was followed by the sequential activation of mitogen-activated protein kinase kinase kinase (MAPKKK) activity of Raf-1, MAP kinase kinase (MAPKK), MAPKs (p44mapk and p42mapk, also called extracellular signal-regulated protein kinase [ERK]1 and ERK2, respectively), and S6 kinase (p90rsk). In this study, we demonstrated that both hypoxia and hypoxia/reoxygenation caused rapid activation of stress-activated MAPK signaling cascades involving p65PAK, p38MAPK, and SAPK. These stimuli also caused phosphorylation of activating transcription factor (ATF)-2. Because p65PAK is known to be upstream of p38MAPK and also be a target of p21rac-1, which belongs to the rho subfamily of p21ras-related small GTP-binding proteins, these results strongly suggested that two different stress-activated MAPK pathways distinct from the classical MAPK pathway were activated in response to hypoxia and hypoxia/reoxygenation in cardiac myocytes.