The pleiotropic drug resistance network in budding yeast presents a first line of defense against xenobiotics, which is formed by primary and secondary active membrane transporters. Among these transporters, the ABC transporter Pdr5 is a key component, because it confers resistance against a broad spectrum of such cytotoxic agents. Furthermore, it represents a model system for homologous transporters from pathogenic fungi and has been intensively studied in the past. In addition to other mutational studies, the S1360F mutation of Pdr5 was found to modulate substrate specificity and resistance. Notably, in the S1360F background, the resistance against the immunosuppressant FK506 is drastically increased. We present a detailed analysis of this mutation that is located in the predicted cytosolic part of transmembrane helix 11. Our data demonstrate that kinetic and thermodynamic parameters of the S1360F mutant are similar to those of the wild-type protein, except for FK506-inhibited ATPase activity and the degree of competitive inhibition. In summary, our results indicate that the S1360F mutation within the transmembrane domain interferes drastically with the ability of the nucleotide-binding domains to hydrolyze ATP by interfering with interdomain crosstalk.