Glucagon increases contractility of the heart muscle by stimulation of adenylate cyclase activity and elevation of cAMP. We have investigated the specific time of onset of glucagon sensitivity of heart muscle during development of the chick embryo. Using both isolated heart preparation and cultured cardiac cells, we have found that the contractile response to glucagon cannot be detected prior to Day 4 of development. Binding studies, carried out with heart cells prepared from 3-, 5-, 7-, and 11-day chick embryos, showed a significant increase in the number of glucagon binding sites between Days 3 and 5. Scatchard analysis showed that for Day 5 cells maximum binding capacity was 0.56 pmole/mg of protein with Kd of 16.0 nM, while for Day 3, maximal binding was only 0.16 pmole/mg with Kd of 15.1 nM. Therefore in this 2-day interval there was a marked increase in the receptor number, without changes in the receptor affinity. Since hormonal stimulation of adenylate cyclase depends on the presence of the regulatory component (Ns), we have used cholera toxin-induced chronotropic effect as an assay for functional Ns. No response to cholera toxin could be detected prior to Day 4 of chick heart development. Therefore, emergence of the cholera toxin sensitivity correlates well with the onset of responsiveness to glucagon. We conclude that as the heart develops it acquires a physiological responsiveness to glucagon. The acquisition of the hormonal sensitivity correlates with the increase in the receptor number and the functional levels of regulatory component.