Mutations in Kir6.2, the pore-forming subunit of the KATP channel, that reduce the ability of ATP to block the channel cause neonatal diabetes. The stimulatory effect of MgATP mediated by the regulatory sulphonylurea receptor (SUR) subunit of the channel may also be modified. We compared the effect of the Kir6.2-F333I mutation on KATP channels containing SUR1, SUR2A or SUR2B. The open probability of Kir6.2/SUR1 channels, or a C-terminally truncated form of Kir6.2 expressed in the absence of SUR, was unaffected by the mutation. However, that of Kir6.2/SUR2A and Kir6.2/SUR2B channels was increased. In the absence of Mg2+, ATP inhibition of all Kir6.2-F333I/SUR channel types was reduced, although SUR1-containing channels were reduced more than SUR2-containing channels. These results suggest F333 is involved in differential coupling of Kir6.2 to SUR1 and SUR2. When Mg2+ was present, ATP blocked SUR2A channels but activated SUR2B and SUR1 channels. Activation by MgGDP (or MgADP) was similar for wild-type and mutant channels and was independent of SUR. This indicates Mg-nucleotide binding to SUR and the transduction of binding into opening of the Kir6.2 pore are unaffected by the mutation. The data further suggest that MgATP hydrolysis by the nucleotide-binding domains of SUR1 and SUR2B, but not SUR2A, is enhanced by the F333I mutation in Kir6.2. Taken together, our data suggest the region of the C terminus within which F333 lies is involved in more than one type of functional interaction with SUR, and that F333 interacts differentially with SUR1 and SUR2.