Cardiac fibroblasts contribute to multiple aspects of myocardial function and pathophysiology. The pathogenetic relevance of cytokine production by these cells under hypoxia, however, remains unexplored. Using an in vitro cell culture model, this study evaluated cytokine production by hypoxic cardiac fibroblasts, and examined two distinct effects of hypoxic fibroblast-conditioned medium on cardiac myocytes and fibroblasts. Hypoxia caused a marked increase in the production of TNF- by cardiac fibroblasts. Hypoxic fibroblast-conditioned medium (HFCM) significantly enhanced the susceptibility of cardiac myocytes to reactive oxygen species (ROS)-induced mitochondrial permeability transition (MPT), determined by high precision confocal line-scan imaging following controlled, photoexcitation-induced ROS production within individual mitochondria. Further, exposure of cardiac myocytes to HFCM for 5 hours led to loss of viability, as evidenced by change in morphology and annexin staining. HFCM also decreased DNA synthesis in cardiac fibroblasts. Normoxic fibroblast-conditioned medium (NFCM) spiked with TNF- at 200 pg/ml, a concentration comparable to that in HFCM, promoted loss of myocyte viability and decreased DNA synthesis in cardiac fibroblasts. These effects of HFCM are similar to the reported effects of hypoxia per se on these cell types, showing that hypoxic fibroblast-derived factors may amplify the distinct effects of hypoxia on cardiac cells. Importantly, since both hypoxia and oxidant stress prevail in a setting of ischemia and reperfusion, the effects of soluble factors from hypoxic fibroblasts on the MPT-ROS threshold and viability of myocytes may represent a novel paracrine mechanism that could exacerbate ischemia-reperfusion injury to cardiomyocytes.