The high-affinity binding of [3H]ouabain to Na+,K+-ATPase was characterized in primary cultures of hippocampal neurons grown on feeder layers of astrocytes. The specific binding of [3H]ouabain was found to be time-dependent, high-affinity (apparent Kd = 8.5 nM), saturable (Bmax = 20.6 pmol/mg protein), dependent upon the presence of ATP, inhibited by K+, and directly proportional to neuronal, but not glial, cell number. Similar results were obtained using either sonicated cell suspensions or intact whole cells in culture. At the concentration of neurons routinely used, the specific binding of [3H]ouabain to the astrocyte feeder layer constituted less than 10% of total specific binding. Agents that selectively kill neurons rather than glia, such as the excitotoxins N-methyl-d-aspartate (NMDA) and kainate, reduced the amount of [3H]ouabain specifically bound in mixed cultures in a time- and concentration-dependent manner. Measurement of high-affinity [3H]ouabain binding to the neuronal form of Na+,K+-ATPase provides a simple, rapid, and reproducible method to quantify neurons in mixed culture.