Cyclic nucleotide-gated (CNG) channels in rod photoreceptors transduce a decrease in cGMP into hyperpolarization during the light response. Insulin-like growth factor-1 (IGF-1) increases light responses by increasing the cGMP sensitivity of CNG channels, an event mediated by a protein tyrosine phosphatase. Native rod CNG channels are heteromultimers, composed of three CNGA1 subunits and one CNGB1 subunit. Previous studies on heterologously expressed rod CNG channels show that a specific tyrosine in the CNGA1 subunit (Y498) is required for modulation by protein tyrosine phosphatases, protein tyrosine kinases and IGF-1. Here we show that the CNGB1 subunit contains a specific tyrosine (Y1097) that is important for modulation of heteromeric channels by tyrosine phosphorylation. Direct biochemical measurements demonstrate 32P-labelling of CNGA1Y498 and CNGB1Y1097. Replacement of either Y498 of CNGA1 or Y1097 of CNGB1 with phenylalanine reduces modulation, and removal of both tyrosines eliminates modulation. Unlike CNGA1, CNGB1 does not exhibit activity dependence of modulation by tyrosine phosphorylation. Hence both CNGA1 and CNGB1 subunits contribute to phosphorylation-dependent modulation of rod CNG channels, but the phosphorylation states of the two subunits are regulated in different ways.