In addition to functioning as a cAMP-activated chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in conferring regulatory properties on other ion channels. It is known, with respect to CFTR regulation of ROMK2 (renally derived K(ATP) channel), that the first transmembrane domain and the first nucleotide binding fold domain (NBF1) of CFTR are necessary for this interaction to occur. It has been shown that under conditions that promote phosphorylation, the ROMK2-CFTR interaction is attenuated. To elucidate the complex nature of this interaction, CFTR constructs were co-expressed with ROMK2 in Xenopus oocytes, and two microelectrode voltage clamp experiments were performed. Although the second half of CFTR can act as a functional chloride channel, our results suggest that it does not confer glibenclamide sensitivity on ROMK2, as does the first half of CFTR. The attenuation of the ROMK2-CFTR interaction under conditions that promote phosphorylation is dependent on at least the presence of the R domain of CFTR. We conclude that transmembrane domain 1, NBF1, and the R domain are the CFTR domains involved in the ROMK2-CFTR interaction and that NBF2 and transmembrane domain 2 are not essential. Lastly, the R domain of CFTR is necessary for the attenuation of the ROMK2-CFTR interaction under conditions that promote phosphorylation.