The Na,K-ATPase is vital for maintenance of lens transparency. Past studies using intact lens suggested the involvement of tyrosine kinases in short-term regulation of Na,K-ATPase. Furthermore, in vitro phosphorylation of a lens epithelial membrane preparation by Src family kinases (SFKs), a family of nonreceptor tyrosine kinases, resulted in modification of Na,K-ATPase activity. Here, the effect of purinergic agonists, ATP and UTP, on Na,K-ATPase function and SFK activation was examined in rabbit lens. Na,K-ATPase function was examined using two different approaches, measurement of ouabain-sensitive potassium (⁸⁶Rb) uptake by the intact lens and Na,K-ATPase activity in lens epithelial homogenates. ATP and UTP caused a significant increase in ouabain-sensitive potassium (⁸⁶Rb) uptake. Na,K-ATPase activity was increased in the epithelium of lenses pre-treated with ATP. Lenses treated with ATP or UTP displayed activation of SFKs as evidenced by increased western blot band density of active SFK (phosphorylated at the active loop Y416) and decreased band density of inactive SFKs (phosphorylated at the C-terminal). A single PY416-Src immunoreactive band at ~60kDa was observed, suggesting not all Src family members are activated. Immunoprecipitation studies showed that band density of active Src, and to a lesser extent active Fyn, was significantly increased while active Yes did not change. Pre-incubation of the lenses with SFK inhibitor PP2 abolished the ATP-induced increase in ouabain-sensitive potassium (⁸⁶Rb) uptake. The results suggest selective activation of Src and/or Fyn is part of a signaling mechanism initiated by purinergic agonists that increases Na,K-ATPase-mediated transport in the organ-cultured lens.