Tetratricopeptide repeat (TPR) domains are ubiquitous structural motifs that mediate protein–protein interactions. For example, the TPR domains in the peroxisomal import receptor PEX5 enable binding to a range of type 1 peroxisomal targeting signal motifs. A homolog of PEX5, tetratricopeptide repeat– containing Rab8b-interacting protein (TRIP8b), binds to and functions as an auxiliary subunit of hyperpolarization-activated cyclic nucleotide (HCN)– gated channels. Given the similarity between TRIP8b and PEX5, this difference in function raises the question of what mechanism accounts for their binding specificity. In this report, we found that the cyclic nucleotide– binding domain and the C terminus of the HCN channel are critical for conferring specificity to TRIP8b binding. We show that TRIP8b binds the HCN cyclic nucleotide– binding domain through a 37-residue domain and the HCN C terminus through the TPR domains. Using a combination of fluorescence polarization– and co-immunoprecipitation– based assays, we establish that binding at either site increases affinity at the other. Thus, allosteric coupling of the TRIP8b TPR domains both promotes binding to HCN channels and limits binding to type 1 peroxisomal targeting signal substrates. These results raise the possibility that other TPR domains may be similarly influenced by allosteric mechanisms as a general feature of protein–protein interactions.