The temporal pattern of changes in the specific activities of retinal Na+, K+-ATPase (Na, K-ATPase) and Mg2+-ATPase (Mg-ATPase) were determined at several time intervals following the onset of diabetes in streptozotocin-induced diabetic (STZ: at 1,2, 4 and 6 months) Long-Evans hooded rats, spontaneously diabetic Zucker diabetic fatty (ZDF: at 1,2 and 4 months) rats and their age-matched controls. These animals were utilized as models for insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM), respectively. Na, K-ATPase specific activity, using 103M ouabain, was decreased (-6% to -14%) at all time points after the appearance of hyperglycemia in the ZDF rat, but was reduced only after 4 and 6 months in the STZ rat (-8% and -14%, respectively). In contrast, Mg-ATPase activity was significantly increased (13%) after 4 months in the ZDF rat and after 6 months in the STZ rat (8%). The concentration-dependent inhibitory effects of ouabain (10-9to 10-3M) on the activity of Na, K-ATPase in diabetic rats and age-matched controls was used to assess the time-dependent effects of diabetes on the $aL3-high ouabain affinity or the $aL1-low ouabain affinity retinal Na, K-ATPase isozymes. The retinal Na, K-ATPase activity for the $aL3 isozyme was significantly lower at all times examined for the ZDF (-5% to -26%) and STZ-induced diabetic rats (-8% to -14%). This was reflected in the markedly decreased half-maximal inhibitory concentrations (IC50) of ouabain for the $aL3 isozyme. For example, after four months of diabetes, the mean±SEMIC50values were 12±3 nM in the STZ rats and 48±6 nM in the age-matched controls and 19±3 nM in the ZDF rats and 30±4 nM in the age-matched controls. In contrast, the activity of the $aL1 isozyme was slightly, but significantly, decreased at 2 and 4 months in the ZDF rats (-4% to -7%) and after 4 and 6 months in the STZ-induced diabetic rats (-3% to -9%) while the IC50values were unchanged. Moreover, the Hill coefficient for the $aL3 isozyme was decreased in both diabetic groups while it was unchanged for the $aL1 isozyme. Our findings suggest that diabetes-induced changes in specific activity and ouabain sensitivity of Na, K-ATPase isozymes result mainly from alterations in the $aL3 isozyme which is located primarily in the outer or distal retina. The exact cellular mechanisms accounting for these changes and their significance in the pathogenesis of diabetic retinopathy remain to be clarified.