The plasma membrane ATP-binding cassette (ABC) transporter, Pdr5p, mediates resistance to many different xenobiotic compounds in yeast. We have isolated several mutated forms that fail to confer resistance to cycloheximide and itraconazole. Here, we examined two variants, the expression of which was abnormally low when cells reach the stationary phase of growth. The Pdr51157 variant lacked the C-terminal transmembrane domain due to the presence of a nonsense mutation at codon 1158. The second variant, Pdr5L183P, contained a Leu183Pro substitution close to the Walker A motif in the N-terminal nucleotide-binding domain. This substitution impaired UTPase activity as well as protein stability. The Pdr5L183P variant induced the unfolded protein response and was targeted to the proteasome for degradation. Fluorescence microscopy showed that the highly unstable Pdr5L183P was mislocalized to endoplasmic reticulum (ER)-associated compartments, whereas the truncated Pdr51157 protein was retained in the ER. When threonine 363 (located in the first nucleotide-binding domain, close to the Walker B motif) in Pdr5L183P was replaced with isoleucine, this double mutant conferred partial drug resistance. These results suggest that Pdr5p requires a properly folded nucleotide-binding domain for trafficking to the plasma membrane.