Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress and H₂O₂ potentiates the concomitant release of the organic osmolyte taurine (Lambert, JMB 192: 19-32, 2003 (35)). The increase in ROS production (carboxy-H2DCFDA fluorescence) is detectable after a reduction in the extracellular osmolarity from 335 mOsm (isotonic) to 300 mOsm and reaches a maximal value after a reduction to 260 mOsm. The swelling-induced ROS production is reduced by the flavoprotein inhibitor diphenylene iodonium chloride (DPI, 25 µM) but unaffected by the nitric oxide synthase inhibitor L-NAME, indicating that the volume-sensitive ROS production is NADPH oxidase dependent. NIH3T3 cells express the NADPH oxidase components: p22^phox, a NOX4 isotype, p47^phox, and p67^phox (RT-PCR). Exposure to the Ca²⁺ mobilizing agonist ATP (10 µM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid-messenger lysophosphatidic acid (LPA, 10 nM) potentiates the swelling-induced taurine release as well as the ROS production. Over-expression of Rac1 or p47^phox or p47^phox knock down (siRNA) had no effect on the swelling-induced ROS production or taurine release. NOX4 knock down (siRNA) impairs the increase in the ROS production and the concomitant taurine release following osmotic exposure. It is suggested that a NOX4 isotype plus p22^phox account for the swelling-induced increase in the ROS production in NIH3T3 cells and that the oxidase activity is potentiated by PKC and LPA but not by Ca²⁺.