High molecular forms of acetylcholinesterase (AChE) have been difficult to obtain in primary cultures of muscle from mammals and birds. This research used pectoral muscle cultures from Japanese quail and chick embryos grown in medium containing embryo extract and horse serum to study the growth and extraction conditions necessary for study of high molecular weight 20S AChE forms found in these birds. The results confirmed the presence of a 20S AChE form in quail muscle cultures and showed that its extraction from the cells was considerably improved by using a Mg-cholate extraction buffer rather than the more commonly used NaCl-Triton X buffer. A striking finding was that removal of serum from the medium for 1-2 days caused the preferential increase in a 20S form in the quail and the resolution of one from background activity in chick muscle cultures. Removal of embryo extract had no effect on levels of the AChE forms. The results suggest that serum factors inhibit the formation of the high molecular weight, motor end plate associated form of AChE in aneural cultures of avian muscle, and that effects of factors such as neural extracts and nerves on AChE forms in cultured muscle should be examined using a defined basal media.