Coexpression of the βwt and αwt subunits of the bovine rod channel restores two characteristics of the native channels: higher sensitivity to cAMP and potentiation of cGMP-induced currents by low cAMP concentrations. To test whether the increased sensitivity to cAMP is due to the uncharged nature of the asparagine residue (N1201) situated in place of aspartate D604 in the β subunit as previously suggested (Varnum et al., 1995, Neuron. 15:619–625), we compared currents from wild-type (αwt and αwt/βwt) and from mutated channels (αD604N, αD604N/βwt, and αwt/βN1201D). The results show that the sensitivity to cAMP and cAMP potentiation is partly but not entirely determined by the charge of residue 1201 in the β subunit. The D604N mutation in the α subunit and, to a lesser extent, coexpression of the βwt subunit with the αwt subunit reduce the open probability for cGMP compared to that of the αwt channel. Interpretation of the data with the MWC allosteric model (model of Monod, Wyman, Changeux; Monod et al., 1965, J. Mol. Biol. 12:88–118) suggests that the D604N mutation in the α subunits and coassembly of α and β subunits alter the free energy of gating by cAMP more than that of cAMP binding.