The development of acetylcholinesterase (AChE) activity in primary surface cultures of mouse cortical neurons and in mouse brain was examined. The specific activity of AChE in culture increased over 600% during a 3 week period and closely paralleled the development of AChE observed in vivo. The results obtained in this study show that a developmental increase in AChE can be obtained in primary surface neuron cultures, and that the high degree of cellular organization previously deemed necessary for this development in vitro is not as important as previously thought.