Mouse brain cell reaggregates have been used to study changes in sodium- and potassium-dependent ouabain-sensitive adenosine phosphohydrolase (Na+, K+-ATPase) activity and in 86Rb+ uptake and exit during development. Na+, K+-ATPase activity in these cultures has two ouabain-inhibitable components, both of which increased severalfold between day 3 and day 17 in culture. This increase, however, was less than that in developing brain. Little change in either total or extracellular water or in the equilibrium levels of Na+ and K+ occurred during development. The uptake of 86Rb+ measured a 10-min incubation showed only a modest increase during culture, whereas the exit of 86Rb+ from reaggregates preloaded with the tracer increased approximately fourfold. The exit consisted of both K+-independent and K+-stimulated components and the K+-stimulated fraction contributed most of the developmental change. When uptake rates were corrected for the contribution of the developmental changes in exit, these rates were found to increase as well. The 86Rb+ uptake correlated closely with the activity of the Na+,K+-ATPase during development. The pattern of developmental changes in enzyme activity and 86Rb+ uptake and exit suggest that, while little change in the steady-state levels of the ions occurred, the rates of ion movement increase markedly.