Several lines of evidence including reverse transcription polymerase chain reaction, immunoreactivity and their ability to efflux rhodamine 123 have implied the existence of P-glycoprotein in natural killer (NK) cells. It has been a natural tendency to assume that NK-cell P-glycoprotein is identical to the P-glycoprotein of multidrug resistant (MDR) cell lines, however, the present study uncovered major differences. Functionally, NK cells demonstrated a restricted substrate profile, being unable to transport daunorubicin and calcein acetoxymethylester while efficiently transporting other P-glycoprotein substrates. Furthermore, physical differences in NK-cell P-glycoprotein were established by differential reactivity with P-glycoprotein antibodies. NK cells demonstrated strong reactivity with C494 and JSB-1, but did not react appreciably with C219. In addition, NK cells were unable to bind to the antibody MM4·17 unless they had been fixed and permeabilized, yet this antibody normally recognizes an extracellular epitope of P-glycoprotein. These differences culminated in the demonstration using Western analysis that NK cells did not express detectable levels of 170 kDa P-glycoprotein. Instead, NK cells expressed small-molecular-weight ‘mini P-glycoprotein’ products, of approximately 70 and 80 kDa. Collectively, these data indicate that the predominant P-glycoprotein species of NK cells are novel mini P-glycoproteins and not the classic P-glycoprotein of MDR models.