The possible relationship between methoxyverapamil (D600) as a calcium-channel blocker and the β-adrenoceptors was investigated on heart cells grown in culture, using [3H]CGP-12177 as a radioligand. Treatment with D600 (20 μg/mL) for 24 hr caused a decrease of 30% in the [3H]CGP-12177 binding sites. Scatchard analysis showed that the Bmax is similar in control and D600-treated cells, but the Kd in D600-treated cells increases. The effect of D600 on the isoproterenol-induced adenylate cyclase activation was examined and it was found that the D600 prevented the increase in cAMP obtained by isoproterenol treatment. These results indicate that the action of D600 on the β-adrenoceptors is a competitive inhibition of the [3H]CGP-12177 binding sites. We investigated the effect of Ca2+ in the growth medium on the level of β-adrenoceptors. Heart cells grown for 24 hr in Ca2+-free medium showed a decrease of 36% in the [3H]CGP-12177 binding sites without changing the dissociation constant. This decrease is probably a result of reduction in synthesis of the receptors. The level of receptors returned to control values following replenishment with normal growth medium. These results show that calcium is essential for the development of the β-adrenoceptors in heart cells in vitro.