The maximal velocity (Vmax) and the apparent dissociation constant (K0.5) of Na+K+-ATPase have each been estimated with respect to sodium and potassium ion activation. These estimations were made from the enzymatic activity of plasma membrane preparations derived from bovine corneal endothelial cells. The determinations were made on cells obtained from fresh tissue and from secondary tissue cultures. Two methods were used to obtain the estimates: the first used a combination of Eadle-Hofstee and Hill plots; the second used Eisenthal-Cornish-Bowden plots. The Vmaxfor sodium was 5.58–5.60μmoles Pj/mg protein/30 min for fresh tissue vs. 2.00–1.80μmoles Pi/mg protein/30 min for tissue cultures. The corresponding K0.5values were 62–57 mM (fresh tissue) vs. 7.9–6.7 mM (tissue culture). Vmaxfor potassium was 4.28–4.00μmoles Pi/mg protein/30 min for fresh tissue vs. 1.37–1.34μmoles Pi/mg protein/30 min for tissue cultures. The corresponding Ko.5 values were 3.3–3.1 mM (fresh tissue) and 1.7–1.7 mM (tissue culture). The results indicate a lowered activity and change in affinity of the enzyme for the two ions in tissue cultures compared to fresh tissues. The detergent Lubrol W-X increased activity in both tissue sources. Sonication had no significant effect on the activity. Variations in pH (7–9) indicated that the highest activity was obtained at pH 7.8 for the enzyme in tissue culture while activity was highest at pH 8.0 for the enzyme in fresh tissues. These differences in kinetic activity suggest a response to changes in the ion requirements of these cells due to their environment, developmental stage or some other parameter.