Both hyaluronidase and transforming growth factor (TGF)-1 play a significant role in the development of prostate cancer. In this study, the regulation of tumor necrosis factor (TNF)-mediated cell death by hyaluronidase and TGF-1 was investigated. Preexposure of L929 fibroblasts, prostate LNCaP cells, and epithelial Mv 1 Lu cells to hyaluronidase for a minimum of 12 h resulted in significant enhancement of cell death by TNF. Phosphorylation of p42 and p44 mitogen-activated protein (MAP) kinases was found by stimulation of L929 cells with hyaluronidase for 30 min, indicating that the Raf/MAP kinase-extracellular signal-regulating protein kinase (MEK)/MAP kinase pathway was activated. However, blocking the activation of upstream MAP kinase kinase (MEK 1 and 2 kinase) by PD-98059 failed to inhibit the hyaluronidase-enhanced TNF killing of cells, suggesting that hyaluronidase-mediated degradation of extracellular matrix and membrane components may elicit multiple signaling pathways. As a potent stimulator of extracellular matrix protein synthesis, TGF-1 blocked the hyaluronidase-enhanced death of L929 and LNCaP cells mediated by TNF. TGF-1 activated protein-tyrosine kinases in L929 cells, in which the tyrosine kinase inhibitors lavendustin A and tyrphostin blocked the activation as well as the TGF-1 inhibition of hyaluronidase effects. Functional antagonism was also observed between hyaluronidase and TGF-1 in cell growth regulation. For example, TGF-1-mediated suppression of epithelial Mv 1 Lu cell growth was abolished by hyaluronidase. Overall, it is demonstrated in this study that hyaluronidase reciprocally antagonized TGF-1 in the modulation of cell proliferation and TNF-mediated death.