Culture of Opossum kidney (OK) cells over 40 passages resulted in marked overexpression of ₁-subunit and ₁-subunit Na⁺,K⁺-ATPase, which paralleled with increases in Na⁺,K⁺-ATPase activity, and enhanced availability in hydrogen peroxide (H₂O₂) (J. Membr. Biol., 212, 163-175, 2006). The present study evaluated the mechanisms involved in increased H₂O₂ production in OK cells cultured over 40 passages (up to passage 80) and whether H₂O₂ plays a role in the regulation of Na⁺,K⁺-ATPase expression and activity. The accumulation of H₂O₂ in the extracellular medium over 24 h (236.5±2.7 vs 319.3±2.9 nM) and the rate of H₂O₂ production (5.21±0.02 vs 7.37±0.02 nM/min) in cells with 40 passages was lower than in cells with 80 passages. The increase in H₂O₂ production in cells with 80 passages was accompanied by overexpression of NOX1, but not of NOX2, and overexpression of SOD1, SOD2 and SOD3. Apocynin, a NADPH inhibitor, markedly attenuated the increased in H₂O₂ production in cells with 80 passages and significantly decreased Na⁺,K⁺-ATPase activity (26% reduction) and ₁-subunit Na⁺,K⁺-ATPase overexpression. Increases in Na⁺,K⁺-ATPase ₁ subunit mRNA abundance were also observed in OK cells with increased number of cell passages, but apocynin did not affect transcript abundance. In conclusion, the increased availability of H₂O₂ in the intracellular milieu plays an important role in the long-term regulation of Na⁺,K⁺-ATPase expression and activity. These cells may constitute an interesting model to study events related to oxidative stress in which adaptation to increases in H₂O₂ had progressively adapted in a more natural manner.