The molecular forms of acetylcholinesterase (AcChE) have been studied in primary cultures of newborn rat sympathetic neurons grown either in the absence (CM- cultures) or in the presence (CM+ cultures) of muscle conditioned medium. The cultures were treated with a mitotic poison to eliminate non-neuronal cells. CAT activity increased with time in culture 4- to 20-fold faster in CM+ than in CM- cultures. In agreement with previous experiments (J.P. Swerts, A. Le Van Thaï, A. Vigny, and M.J. Weber, 1983, Develop. Biol. 100, 1-11), AcChE activity developed at a 3-fold lower rate in CM+ than in CM- cultures. This deficit in AcChE activity in CM+ cultures resulted from a deficit in the number of enzyme molecules immunoprecipitable with an antiserum raised against rat brain AcChE. In both types of cultures, AcChE forms were separated by sucrose gradient sedimentation into three main peaks corresponding to the 16 S and 10 S forms and a mixture of the 6.5 and 4 S forms. In 3-day-old CM+ and CM- cultures, the 16 S form represented 2% of the total activity. After 12-26 days, the percentage of 16 S form raised to 15-30% in CM- cultures, but remained lower than 5% in CM+ cultures. This difference was also observed when AcChE molecular forms were analyzed in the presence of protease inhibitors. A similar result was obtained by comparing cultures grown with and without a macromolecular factor partially purified from conditioned medium. These results suggest that an inverse relationship exists between the presence of 16 S AcChE and the presence of cholinergic synapses in these cultures.