Primary culture of chick heart cells has proven useful in biochemical and physiological investigations of receptor-mediated myocardial responses. While cell culture provides a system in which the cellular environment can be manipulated in carefully controlled ways, the use of serum as a culture medium supplement introduces variability due to the inherent differences in the composition of different lots of sera. In this report we compare the functional properties of the muscarinic acetylcholine receptor of cultured embryonic chick heart cells grown in serum-free, defined medium to that of cells cultured in serum-containing medium. We demonstrate that heart cells cultured in defined medium express the muscarinic receptor at similar levels and with similar affinity for both agonists and antagonists as do cells cultured in the presence of serum. In addition, we show that heart cells cultured in defined medium respond to the muscarinic agonist carbachol with a loss of muscarinic receptor number, inhibition of cAMP accumulation, and stimulation of phosphoinositide breakdown. Responses were of similar or greater magnitude in cells cultured in defined medium than those obtained from cells grown in serum-containing medium. These results indicate that chick heart cells can be cultured in serum-free, defined medium with retention of muscarinic receptor function. Thus, this culture system should be useful for the study of the regulation of muscarinic receptor function by hormones, growth factors and other serum components without the complications caused by serum-supplemented media.