The development of the molecular forms of acetylcholinesterase was studied in rat heart during the perinatal period. In this study, the activity of acetylcholinesterase increased both per unit wet weight and per unit protein from post-conception day 14 to day 42. Additionally, the activity of atrial acetylcholinesterase per unit wet weight increased more rapidly after birth than that of ventricular acetylcholinesterase. The percent contribution of the various molecular forms to the total acetylcholinesterase pool in heart changed dramatically from the fetal to the neonatal period. This switch primarily consisted of a decrease in the ratio of the asymmetric to globular forms. Thus, the specific activity of globular forms increased while that of the asymmetric forms remained relatively stable. When the atria and ventricles were examined separately at 19 days post-conception, the percent contribution of the individual molecular forms in the two cardiac areas was different. The atria contained a pool of acetylcholinesterase forms similar to postnatal heart while the ventricles contained a pool of acetylcholinesterase forms with a lower globular to asymmetric ratio. Finally, this study showed that greater than half of the acetylcholinesterase pool was inhibited by incubating hearts from fetal rats with echothiopate iodide, suggesting that a large portion of acetylcholinesterase catalytic sites are externalized in fetal heart.