Assays for complete quantification of Na+, K+-and Ca2+-ATPase in crude homogenates of rat ventricular myocardium by determination of K+-and Ca2+-dependent p-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K+-and Ca2+-dependent pNPPase activities in ventricular myocardium of 11-12 week-old rats were found to be 2.98±0.10 and 0.29±0.02 μmol×min-1×g-1 wet wt. (mean±SEM) (n=5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na+, K+-and Ca2+-ATPase concentrations were estimated to 2 and 10 nmol×g-1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca2+-dependent pNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K+-dependent pNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple out-come of variations in water and protein content of myocardium. Expressed per heart, the K+-and Ca2+-dependent pNPPase activity gradually increased to a plateau. The present assay for Na+, K+-ATPase quantification has the advantage over [3H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K+-dependent 3-0-methylfluorescein phosphatase (3-0-MFPase) in crude tissue homogenates. Furthermore, with few modifications the pNPPase assay allows quantification of Ca2+-ATPase on crude myocardial homogenates. Age-dependent changes in K+-and Ca2+-dependent pNPPase activities are of developmental interest and indicate the importance of close age match in studies of quantitative aspects of Na+, K+-and Ca2+-ATPase in excitable tissues.