The Na/K-exchange characteristics, ouabain-binding kinetics, and Na pump turnover rates of synchronously contracting monolayers of neonatal rat myocardial cells were studied. The cells exchange Na rapidly (T1/2 = 35 s) with a mean Na flux of approximately 25 (pmol/cm2)/s. The half time (T1/2) of K exchange is much longer (12 min); the mean K flux is 13 (pmol/cm2)/s. Active Na/K transport, as measured by K influx, is relatively ouabain sensitive, and 10(-6) M ouabain produces half-maximal inhibition. Ouabain (10(-2)M) inhibits 60% of the Na efflux and 75% of the K influx. The cells bind [3H]ouabain rapidly (T1/2 = 8 min), but release it very slowly (T1/2 = 11 h), and both the amount bound and the rate of binding were inversely proportional to extracellular K. Specific [3H]ouabain binding demonstrates saturation reaching a maximum of 1.6 x 10(6) molecules per cell at 2 x 10(-7) M [3H]ouabain. From cell surface area and ouabain-sensitive flux measurements, the Na pump density was calculated at 720/micrometer2 with an individual pump turnover rate of 50/s. Thus the studies indicate that despite their neonatal origin, the behavior of the Na pump in these cells is very similar to that in other mammalian tissues.